CN116490605A

CN116490605A - Methods of treating sensitive patients with low immunity cells and related methods and compositions

Info

Publication number: CN116490605A
Application number: CN202180067169.7A
Authority: CN
Inventors: S·施雷普费尔; S·哈尔; C·E·玛丽
Original assignee: Sana Biotechnology Co ltd
Current assignee: Sana Biotechnology Co ltd
Priority date: 2020-08-13
Filing date: 2021-08-12
Publication date: 2023-07-25

Abstract

Disclosed herein are low immunity cells for administration to a sensitive patient. In some examples, the patient is sensitive to previous pregnancy or previous grafts. In some embodiments, the cell exogenously expresses CD47 protein and exhibits reduced expression of MHC class I proteins, MHC class II proteins, or both.

Description

Methods of treating sensitive patients with low immunity cells and related methods and compositions

Cross Reference to Related Applications

The present application claims 35U.S. c. ≡119 (e) U.S. provisional application No. 63/065,342 to day 13, 2020; no. 63/136,137 of application Ser. No. 11/1 of 2021; no. 63/151,628 of application No. 19, 2/2021; and priority of application number 63/175,030, 14, 4, 2021, the disclosures of which are incorporated herein by reference in their entirety.

Background

Allergy to antigens (e.g., donor alloantigens) is a problem faced by clinical transplantation therapies. For example, the propensity of the immune system of the graft recipient to repel allogeneic materials greatly reduces the potential efficacy of the treatment and reduces the possible positive impact surrounding such treatment. Fortunately, there is substantial evidence for both animal models and human patients that low immunity cell or tissue transplantation is a scientifically viable and clinically promising approach to treating a variety of disorders and conditions.

Thus, there remains a need for new approaches, compositions and methods for generating cell-based therapies that avoid detection by the immune system of a recipient.

Disclosure of Invention

In some aspects, methods of treating a patient in need thereof are provided comprising administering a population of low-immunity cells, wherein the low-immunity cells comprise a first exogenous polynucleotide encoding CD47 and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: the patient is a sensitive patient, wherein the patient: (i) sensitive to one or more alloantigens; (ii) is sensitive to one or more autoantigens; (iii) sensitive due to previous grafts; (iv) sensitivity due to previous pregnancy; (v) receiving prior treatment for the disorder or disease; and/or (vi) is a tissue or organ transplant patient and the low immunity cells are administered prior to, concurrently with, and/or after administration of the tissue or organ transplant.

In some aspects, methods of treating a patient in need thereof are provided, comprising administering a population of pancreatic islet cells, wherein the pancreatic islet cells comprise a first exogenous polynucleotide encoding CD47 and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: (a) the patient is not a sensitive patient; or (b) the patient is a sensitive patient, wherein the patient: (i) sensitive to one or more alloantigens; (ii) is sensitive to one or more autoantigens; (iii) sensitive due to previous grafts; (iv) sensitivity due to previous pregnancy; (v) receiving prior treatment for the disorder or disease; and/or (vi) is a tissue or organ patient, and the pancreatic islet cells are administered prior to administration of the tissue or organ transplant.

In some aspects, there is provided a method of treating a patient in need thereof, comprising administering a population of cardiac progenitor cells, wherein the cardiac progenitor cells comprise a first exogenous polynucleotide encoding CD47 and (I) one or more of the following: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: (a) the patient is not a sensitive patient; or (b) the patient is a sensitive patient, wherein the patient: (i) sensitive to one or more alloantigens; (ii) is sensitive to one or more autoantigens; (iii) sensitive due to previous grafts; (iv) sensitivity due to previous pregnancy; (v) receiving prior treatment for the disorder or disease; and/or (vi) is a tissue or organ patient and the cardiac muscle cells are administered prior to administration of the tissue or organ transplant.

In some aspects, methods of treating a patient in need thereof are provided, comprising administering a population of glial progenitor cells, wherein the glial progenitor cells comprise a first exogenous polynucleotide encoding CD47 and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: (a) the patient is not a sensitive patient; or (b) the patient is a sensitive patient, wherein the patient: (i) sensitive to one or more alloantigens; (ii) is sensitive to one or more autoantigens; (iii) sensitive due to previous grafts; (iv) sensitivity due to previous pregnancy; (v) receiving prior treatment for the disorder or disease; and/or (vi) is a tissue or organ patient, and prior to administration of the tissue or organ transplant, glial progenitor cells are administered.

In some embodiments, the patient is a sensitive patient, and wherein the patient exhibits a memory B-cell and/or memory T-cell response against one or more alloantigens or one or more autoantigens. In some embodiments, the one or more alloantigens comprise human leukocyte antigens.

In some embodiments, the patient is a sensitive patient that is sensitive to a previous graft, wherein: (a) the prior graft is selected from the group consisting of: cell grafts, blood transfusions, tissue grafts and organ grafts, the previous grafts being allografts as required; or (b) the previous graft is a graft selected from the group consisting of: chimeras of human origin, modified non-human autologous cells, modified autologous cells, autologous tissues and organs, optionally, the previous grafts are autografts.

In some embodiments, the patient is a susceptible patient who is susceptible to prior pregnancy, and wherein the patient has previously exhibited alloimmunity during pregnancy (alloimmunizatio n), optionally wherein the alloimmunity during pregnancy is fetal and neonatal Hemolytic Disease (HDFN), neonatal Alloimmune Neutropenia (NAN), or fetal and neonatal alloimmune thrombocytopenia (FNAIT).

In some embodiments, the patient is a susceptible patient that is susceptible to prior treatment of a disorder or disease, wherein the disorder or disease is different from or the same as the disorder or disease of the patient being treated as described herein.

In some embodiments, the patient receives a prior treatment for the disorder or disease, wherein the prior treatment does not comprise the population of cells, and wherein: (a) Administering the population of cells to treat the same disorder or disease previously treated; (b) The population of cells exhibits an enhanced therapeutic effect on the treatment of a disorder or disease in a patient compared to prior treatments; (c) The population of cells exhibits a longer therapeutic effect on the treatment of a disorder or disease in a patient than prior treatments; (d) the prior treatment is therapeutically effective; (e) prior treatment is treatment ineffective; (f) the patient develops an immune response to the previous treatment; and/or (g) administering the population of cells to treat a condition or disease that is different from the previous treatment.

In some embodiments, the prior treatment comprises administering a population of therapeutic cells comprising a suicide gene or safety switching system, and an immune response occurs in response to activation of the suicide gene or safety switching system.

In some embodiments, the prior treatment comprises a mechanically assisted treatment, optionally, wherein the mechanically assisted treatment comprises hemodialysis or ventricular assist devices.

In some embodiments, the prior treatment comprises an allogeneic CAR-T cell-based therapy or an autologous CAR-T cell-based therapy, wherein the autologous CAR-T cell-based therapy is selected from the group consisting of: bukaba Ji Aolun (brexucabtagene autoleuc el), cekasiro (axicabtagene ciloleucel), ai Kaba Ji Weisai (idecabtagene vicleucel), li Jimai Racing malassezia (lisocabtagene maraleucel), te Sha Jinlu (tis agenlecleucel), descartes-08 or Descarts es-11 from Cartesian Therapeutics, CTL110 from Novartis, P-BM CA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Celectis, PBCAR19B or PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics and CYAD-211 from Clyad Oncology.

In some embodiments, the patient is suffering from allergy, optionally wherein the allergy is allergy hay fever, food allergy, insect allergy, drug allergy, and atopic dermatitis selected from the group consisting of.

In some embodiments, the cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof.

In some embodiments, the cell further comprises a reduced amount of CD142 relative to a cell of the same cell type that does not comprise the modification. In some embodiments, the cell further comprises a reduced amount of CD46 relative to a cell of the same cell type that does not comprise the modification. In some embodiments, the cell further comprises a reduced amount of CD59 relative to a cell of the same cell type that does not comprise the modification.

In some embodiments, the cells differentiate from stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a pluripotent stem cell, optionally, wherein the pluripotent stem cell is an induced pluripotent stem cell. In some embodiments, the cell is selected from the group consisting of: cardiac cells, cardiac progenitor cells, neural cells, glial progenitor cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, chimeric Antigen Receptor (CAR) T cells, NK cells, and CAR-NK cells. In some embodiments, the cells are derived from primary cells. In some embodiments, the primary cell is a primary T cell, a primary β cell, or a primary retinal pigment epithelial cell. In some embodiments, the cells derived from primary T cells are derived from a T cell pool comprising primary T cells from one or more subjects other than the patient.

In some embodiments, the cell comprises a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR). In some embodiments, the antigen binding domain of the CAR binds to CD19, CD22, or BCMA.

In some embodiments, the CAR is a CD 19-specific CAR, such that the cell is a CD19 CAR T cell. In some embodiments, the CAR is a CD 22-specific CAR, such that the cell is a CD22 CAR T cell. In some embodiments, the cell comprises a CD 19-specific CAR and a CD 22-specific CAR, such that the cell is a CD19/CD22 CAR T cell. In some embodiments, the CD 19-specific CAR and the CD 22-specific CAR are encoded by a single bicistronic polynucleotide. In some embodiments, the CD 19-specific CAR and the CD 22-specific CAR are encoded by two separate polynucleotides.

In some embodiments, the first and/or second exogenous polynucleotide is inserted into a genomic locus comprising a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus.

In some embodiments, the first and second genomic loci are the same. In some embodiments, the first and second genomic loci are different. In some embodiments, the cells each further comprise a third exogenous polynucleotide inserted into a third genomic locus. In some embodiments, the third genomic locus is identical to the first or second genomic locus. In some embodiments, the third genomic locus is different from the first and/or second genomic loci.

In some embodiments, the safe harbor locus is selected from the group consisting of: CCR5 gene locus, PPP1R12C (also known as AAVS 1) gene, ROSA26 gene locus and CLYBL gene locus. In some embodiments, the target locus is selected from the group consisting of: CXCR4 locus, albumin locus, SHS231 locus, CD142 locus, MICA locus, MICB locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus.

In some embodiments, the insertion into the CCR5 gene locus is at exons 1 to 3, introns 1 to 2, or another coding sequence (CDS) of the CCR5 gene. In some embodiments, the insertion into the PPP1R12C gene locus is intron 1 or intron 2 of the PPP1R12C gene. In some embodiments, the insertion into the CLYBL gene locus is intron 2 of the CLYBL gene. In some embodiments, the insertion into the ROSA26 gene locus is intron 1 of the ROSA26 gene. In some embodiments, the insertion into the safe harbor locus is the SHS231 locus. In some embodiments, the insertion into the CD142 gene locus is at exon 2 of the CD142 gene or another CDs. In some embodiments, the insertion into the MICA gene locus is at the CDS of the MICA gene. In some embodiments, the insertion into the MICB gene locus is at the CDS of the MICB gene. In some embodiments, the insertion into the B2M gene locus is at exon 2 of the B2M gene or another CDS. In some embodiments, the insertion into the CIITA gene locus is at exon 3 of the CIITA gene or another CDS. In some embodiments, the insertion into the TRAC gene locus is at exon 2 of the TRAC gene or another CDS. In some embodiments, the insertion into the TRB gene locus is at the CDS of the TRB gene.

In some embodiments, the primary T cell-derived cells comprise one or more of the following that reduce expression: endogenous T cell receptors; cytotoxic T-lymphocyte-associated protein 4 (CTLA 4); programmed cell death (PD 1); and programmed cell death ligand 1 (PD-L1), wherein the reduced expression is due to the modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification. In some embodiments, the cells derived from primary T cells comprise reduced expression TRAC.

In some embodiments, the cell is a T cell derived from an induced pluripotent stem cell comprising one or more of the following that reduces expression: endogenous T cell receptors; cytotoxic T-lymphocyte-associated protein 4 (CTLA 4); programmed cell death (PD 1); and programmed cell death ligand 1 (PD-L1). In some embodiments, the cell is a T cell derived from an induced pluripotent stem cell comprising reduced expression of TRAC and TRB.

In some embodiments, the exogenous polynucleotide is operably linked to a promoter. In some embodiments, the promoter is a CAG and/or EF1a promoter.

In some embodiments, the population of cells is administered for at least 1 day or more after the patient is sensitive to one or more alloantigens, or for at least 1 day or more after the patient receives an allograft. In some embodiments, the population of cells is administered for at least one week or more after the patient is sensitive to one or more alloantigens, or at least one week or more after the patient receives an allograft.

In some embodiments, the population of cells is administered for at least 1 month or more after the patient is sensitive to one or more alloantigens, and for at least 1 month or more after the patient receives the allograft.

In some embodiments, the patient exhibits no immune response after administration of the population of cells. In some embodiments, the immune-free response after administration of the population of cells is selected from the group consisting of: no systemic immune response, no adaptive immune response, no innate immune response, no T cell response, no B cell response, and no systemic acute cellular immune response.

In some embodiments, the patient exhibits one or more of the following: (a) After administration of the population of cells, there is no systemic TH1 activation; (b) Immune activation of Peripheral Blood Mononuclear Cells (PBMCs) is absent following administration of the population of cells; (c) After administration of the population of cells, no donor-specific IgG antibodies are directed against the population of cells; (d) Upon administration of the population of cells, no IgM and IgG antibodies are produced to the population of cells; and (e) no cytotoxic T cell killing of the population of cells after administration of the population of cells.

In some embodiments, the patient is not administered the immunosuppressant for at least 3 days or more before or after administration of the population of cells.

In some embodiments, the method comprises a dosing regimen comprising: a first administration comprising a therapeutically effective amount of the population of cells; during recovery; and a second administration comprising a therapeutically effective amount of the population of cells. In some embodiments, the recovery period comprises at least 1 month or more. In some embodiments, the recovery period comprises at least 2 months or more.

In some embodiments, the second administration is initiated when cells from the first administration are no longer detectable in the patient, optionally wherein cells are no longer detectable because of removal from the suicide gene or safety switching system.

In some embodiments, the low immunity cells are removed by suicide genes or safety switching systems, and wherein the second administration is initiated when cells from the first administration are no longer detectable in the patient.

In some embodiments, the method further comprises administering the dosing regimen at least twice. In some embodiments, the population of cells is administered for treating a cell defect or as a cell therapy for treating a disorder or disease in a tissue or organ selected from the group consisting of: heart, lung, kidney, liver, pancreas, intestine, stomach, cornea, bone marrow, blood vessels, heart valves, brain, spinal cord, and bone.

In some embodiments of the method: (a) Cell defects are associated with neurodegenerative diseases or cell therapies for treating neurodegenerative diseases; (b) Cell defects are associated with liver disease or cell therapies for treating liver disease; (c) Cell deficiencies are associated with corneal diseases or cell therapies for treating corneal diseases; (d) Cell deficiencies are associated with cardiovascular disorders or diseases or cell therapies for treating cardiovascular disorders or diseases; (e) Cell deficiencies associated with diabetes or cell therapies for treating diabetes; (f) Cell defects are associated with vascular disorders or diseases or cell therapies are used to treat vascular disorders or diseases; (g) Cell deficiencies are associated with autoimmune thyroiditis or cell therapies for the treatment of autoimmune thyroiditis; or (h) a cell deficiency associated with kidney disease or cell therapy for treating kidney disease.

In some embodiments of the method: (a) a neurodegenerative disease selected from the group consisting of: leukodystrophy, huntington's disease, parkinson's disease, multiple sclerosis, transverse myelitis, and Pemetrexed (PMD); (b) liver disease comprises cirrhosis of the liver; (c) The cornea disease is Fuchs dystrophic (Fuchs dystophy) or congenital genetic endothelial dystrophy; or (d) the cardiovascular disease is myocardial infarction or congestive heart failure.

In some embodiments, the population of cells comprises: (a) a cell selected from the group consisting of: glial progenitor cells, oligodendrocytes, astrocytes and dopamine neurons, optionally, wherein the dopamine neurons are selected from the group consisting of: neural stem cells, neural progenitor cells, immature dopamine neurons, and mature dopamine neurons; (b) hepatocytes or hepatic progenitors; (c) corneal endothelial progenitor cells or corneal endothelial cells; (d) cardiomyocytes or cardiac progenitors; (e) Pancreatic islet cells, including pancreatic beta islet cells, optionally, wherein the pancreatic islet cells are selected from the group consisting of: pancreatic islet progenitor cells, immature pancreatic islet cells, and mature pancreatic islet cells; (f) endothelial cells; (g) thyroid progenitor cells; or (h) a kidney precursor cell or kidney cell.

In some embodiments, the population of cells is administered to treat cancer. In some embodiments, the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

In some embodiments, the patient is receiving a tissue or organ transplant, optionally, wherein the tissue or organ transplant or partial organ transplant is selected from the group consisting of: heart graft, lung graft, kidney graft, liver graft, pancreas graft, intestinal graft, stomach graft, cornea graft, bone marrow graft, vascular graft, heart valve graft, bone graft, partial lung graft, partial kidney graft, partial liver graft, partial pancreas graft, partial intestinal graft, and partial cornea graft.

In some embodiments, the tissue or organ graft is an allograft graft. In some embodiments, the tissue or organ graft is an autograft graft.

In some embodiments, the population of cells is administered to treat a cell defect in a tissue or organ and the tissue or organ transplant is a replacement for the same tissue or organ. In some embodiments, the population of cells is administered to treat a cell defect in a tissue or organ and the tissue or organ transplant is a replacement for a different tissue or organ. In some embodiments, the organ transplant is a kidney transplant and the population of cells is a population of pancreatic beta islet cells. In some embodiments, the patient has diabetes. In some embodiments, the organ transplant is a heart transplant and the population of cells is a population of pacing cells. In some embodiments, the organ transplant is a pancreatic transplant and the population of cells is a population of beta islet cells. In some embodiments, the organ transplant is a partial liver transplant and the population of cells is a population of hepatocytes or hepatic progenitors.

In some aspects, provided herein is a use of a population of low immunity cells for treating a disorder in a patient, wherein the low immunity cells comprise a first exogenous polynucleotide encoding CD47 and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: (a) the patient is not a sensitive patient; or (b) the patient is a sensitive patient.

In some aspects, provided herein is a use of a population of pancreatic islet cells for treating a disorder in a patient, wherein the pancreatic islet cells comprise a first exogenous polynucleotide encoding CD47 and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: (a) the patient is not a sensitive patient; or (b) the patient is a sensitive patient.

In some aspects, provided herein is a use of a population of heart muscle cells for treating a disorder in a patient, wherein the heart muscle cells comprise a first exogenous polynucleotide encoding CD47 and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: (a) the patient is not a sensitive patient; or (b) the patient is a sensitive patient.

In some aspects, provided herein is a method for treating a disorder in a patient, wherein the method comprises administering to the patient a population of glial progenitor cells comprising a first exogenous polynucleotide encoding CD47 and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: (a) the patient is not a sensitive patient; or (b) the patient is a sensitive patient.

In some embodiments, the patient is a sensitive patient that is sensitive to a previous graft, wherein: the previous graft is selected from the group consisting of: cell grafts, blood transfusions, tissue grafts and organ grafts, the previous grafts being allografts as required; or the previous graft is a graft selected from the group consisting of: chimeras of human origin, modified non-human autologous cells, modified autologous cells, autologous tissues and organs, optionally, the previous grafts are autografts.

In some embodiments, the patient is a susceptible patient who is susceptible to prior pregnancy, and wherein the patient has previously exhibited an alloimmune effect during pregnancy, optionally wherein the alloimmune effect during pregnancy is Hemolytic Disease (HDFN) in the fetus and neonate, neonatal Alloimmune Neutropenia (NAN), or fetal and neonatal alloimmune thrombocytopenia (FNAIT).

In some embodiments, the patient is a sensitive patient that is sensitive to the disorder or previous treatment of the disorder. In some embodiments, the patient receives a prior treatment for the disorder or disease, wherein the prior treatment does not comprise the population of cells, and wherein: (a) Administering the population of cells to treat the same disorder or disease previously treated; (b) The population of cells exhibits an enhanced therapeutic effect on the treatment of a disorder or disease in a patient compared to prior treatments; (c) The population of cells exhibits a longer therapeutic effect on the treatment of a disorder or disease in a patient than prior treatments; (d) the prior treatment is therapeutically effective; (e) prior treatment is treatment ineffective; (f) the patient develops an immune response to the previous treatment; and/or (g) administering the population of cells to treat a condition or disease that is different from the previous treatment.

In some embodiments, the cells differentiate from stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a pluripotent stem cell, optionally, wherein the pluripotent stem cell is an induced pluripotent stem cell.

In some embodiments, the cell is selected from the group consisting of: cardiac cells, neural cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, chimeric Antigen Receptor (CAR) T cells, NK cells, and CAR-NK cells. In some embodiments, the cell is derived from a primary cell. In some embodiments, the primary cell is a primary T cell, a primary β cell, or a primary retinal pigment epithelial cell. In some embodiments, the cells derived from primary T cells are derived from a T cell pool comprising primary T cells from one or more subjects other than the patient.

In some embodiments, the cell comprises a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR). In some embodiments, the antigen binding domain of the CAR binds to CD19, CD22, or BCMA. In some embodiments, the CAR is a CD 19-specific CAR, such that the cell is a CD19 CAR T cell. In some embodiments, the CAR is a CD 22-specific CAR, such that the cell is a CD22 CAR T cell. In some embodiments, the cell comprises a CD 19-specific CAR and a CD 22-specific CAR, such that the cell is a CD19/CD22 CAR T cell. In some embodiments, the CD 19-specific CAR and the CD 22-specific CAR are encoded by a single bicistronic polynucleotide. In some embodiments, the CD 19-specific CAR and the CD 22-specific CAR are encoded by two separate polynucleotides.

In some embodiments, the safe harbor locus is selected from the group consisting of: CCR5 gene locus, PPP1R12C (also known as AAVS 1) gene and CLYBL gene locus.

In some embodiments, the target locus is selected from the group consisting of: CXCR4 locus, albumin locus, SHS231 locus, ROSA26 locus, CD142 locus, MICA locus, MICB locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

In some embodiments, the primary T cell-derived cells comprise one or more of the following that reduce expression: (a) an endogenous T cell receptor; (b) Cytotoxic T-lymphocyte-associated protein 4 (CTLA 4); (c) programmed cell death (PD 1); and (d) a programmed cell death ligand 1 (PD-L1), wherein the reduced expression is due to the modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification. In some embodiments, the cells derived from primary T cells comprise reduced expression TRAC.

In some embodiments, the cell is a T cell derived from an induced pluripotent stem cell comprising one or more of the following that reduces expression: (a) an endogenous T cell receptor; (b) Cytotoxic T-lymphocyte-associated protein 4 (CTLA 4); (c) programmed cell death (PD 1); and (d) a programmed cell death ligand 1 (PD-L1), wherein the reduced expression is due to the modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification. In some embodiments, the cell is a T cell derived from an induced pluripotent stem cell comprising reduced expression of TRAC and TRB.

In some embodiments, the population of cells is administered for at least 1 day or more after the patient is sensitive to one or more alloantigens, or for at least 1 day or more after the patient receives an allograft. In some embodiments, the population of cells is administered for at least one week or more after the patient is sensitive to one or more alloantigens, or at least one week or more after the patient receives an allograft. In some embodiments, the population of cells is administered for at least 1 month or more after the patient is sensitive to one or more alloantigens, and for at least 1 month or more after the patient receives the allograft.

In some embodiments, the method comprises a dosing regimen comprising: (a) A first administration comprising a therapeutically effective amount of the population of cells; (b) during recovery; and (c) a second administration comprising a therapeutically effective amount of the population of cells. In some embodiments, the recovery period comprises at least 1 month or more. In some embodiments, the recovery period comprises at least 2 months or more. In some embodiments, the second administration is initiated when cells from the first administration are no longer detectable in the patient.

In some embodiments, the use of the cell further comprises administering the dosing regimen at least twice.

In some embodiments, the population of cells is administered for treating a cell defect or as a cell therapy to treat a disorder or disease in a tissue or organ selected from the group consisting of: heart, lung, kidney, liver, pancreas, intestine, stomach, cornea, bone marrow, blood vessels, heart valves, brain, spinal cord, and bone.

In some embodiments, (a) the cell deficiency is associated with a neurodegenerative disease or cell therapy is used to treat a neurodegenerative disease; (b) Cell defects are associated with liver disease or cell therapies for treating liver disease; (c) Cell deficiencies are associated with corneal diseases or cell therapies for treating corneal diseases; (d) Cell deficiencies are associated with cardiovascular disorders or diseases or cell therapies for treating cardiovascular disorders or diseases; (e) Cell deficiencies associated with diabetes or cell therapies for treating diabetes; (f) Cell defects are associated with vascular disorders or diseases or cell therapies are used to treat vascular disorders or diseases; (g) Cell deficiencies are associated with autoimmune thyroiditis or cell therapies for the treatment of autoimmune thyroiditis; or (h) a cell deficiency associated with kidney disease or cell therapy for treating kidney disease.

In some embodiments, (a) the neurodegenerative disease is selected from the group consisting of: leukodystrophy, huntington's disease, parkinson's disease, multiple sclerosis, transverse myelitis, and Pemetrexed (PMD); (b) liver disease comprises cirrhosis of the liver; (c) The cornea disease is Fuchs dystrophy or congenital genetic endothelial dystrophy; or (d) the cardiovascular disease is myocardial infarction or congestive heart failure.

In some embodiments, the population of cells comprises: (a) a cell selected from the group consisting of: glial progenitor cells, (b) oligodendrocytes, astrocytes and dopamine neurons, optionally, wherein the dopamine neurons are selected from the group consisting of: neural stem cells, neural progenitor cells, immature dopamine neurons, and mature dopamine neurons; (c) hepatocytes or hepatic progenitors; (d) corneal endothelial progenitor cells or corneal endothelial cells; (e) cardiomyocytes or cardiac progenitors; (f) Pancreatic islet cells, including pancreatic beta islet cells, optionally, wherein the pancreatic islet cells are selected from the group consisting of: pancreatic islet progenitor cells, immature pancreatic islet cells, and mature pancreatic islet cells; (g) endothelial cells; (h) thyroid progenitor cells; or (i) a kidney precursor cell or kidney cell.

In some embodiments, the population of cells is administered to treat a cell defect in a tissue or organ and the tissue or organ transplant is a replacement for the same tissue or organ. In some embodiments, the population of cells is administered to treat a cell defect in a tissue or organ and the tissue or organ transplant is a replacement for a different tissue or organ. In some embodiments, the organ transplant is a kidney transplant and the population of cells is a population of kidney precursor cells or kidney cells. In some embodiments, the patient has diabetes. In some embodiments, the organ transplant is a heart transplant and the population of cells is a population of heart progenitor cells or paced cells. In some embodiments, the organ transplant is a pancreatic transplant and the population of cells is a population of pancreatic beta islet cells. In some embodiments, the organ transplant is a partial liver transplant and the population of cells is a population of hepatocytes or hepatic progenitors.

In some aspects, provided herein are methods of treating a patient in need thereof, comprising administering a population of low immunity cells, wherein the low immunity cells comprise a first exogenous polynucleotide encoding CD47, a second exogenous polynucleotide encoding a CAR, and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: (a) the patient is not a sensitive patient; or (b) the patient is a sensitive patient, wherein the patient: (i) sensitive to one or more alloantigens; (ii) is sensitive to one or more autoantigens; (iii) sensitive due to previous grafts; (iv) sensitivity due to previous pregnancy; (v) receiving prior treatment for the disorder or disease; and/or (vi) is a tissue or organ patient and the low immunity cells are administered prior to administration of the tissue or organ transplant.

In some embodiments, the cells differentiate from stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a pluripotent stem cell, optionally, wherein the pluripotent stem cell is an induced pluripotent stem cell. In some embodiments, the cell is a CAR T cell or CAR-NK cell. In some embodiments, the cells are derived from primary T cells. In some embodiments, the cells are derived from a T cell pool comprising primary T cells from one or more subjects other than the patient.

In some embodiments, the antigen binding domain of the CAR binds to CD19, CD22, or BCMA. In some embodiments, the CAR is a CD 19-specific CAR, such that the cell is a CD19 CAR T cell. In some embodiments, the CAR is a CD 22-specific CAR, such that the cell is a CD22CAR T cell. In some embodiments, the cell comprises a CD 19-specific CAR and a CD 22-specific CAR, such that the cell is a CD19/CD22CAR T cell. In some embodiments, the CD 19-specific CAR and the CD 22-specific CAR are encoded by a single bicistronic polynucleotide.

In some embodiments, the CD 19-specific CAR and the CD 22-specific CAR are encoded by two separate polynucleotides.

In some embodiments, the first and/or second exogenous polynucleotide is inserted into a genomic locus comprising a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus. In some embodiments, the first and second genomic loci are the same. In some embodiments, the first and second genomic loci are different.

In some embodiments, the cells each further comprise a third exogenous polynucleotide inserted into a third genomic locus. In some embodiments, the third genomic locus is identical to the first or second genomic locus. In some embodiments, the third genomic locus is different from the first and/or second genomic loci.

In some embodiments, the safe harbor locus is selected from the group consisting of: CCR5 gene locus, PPP1R12C (also known as AAVS 1) gene and CLYBL gene locus. In some embodiments, the target locus is selected from the group consisting of: CXCR4 locus, albumin locus, SHS231 locus, ROSA26 locus, CD142 locus, MICA locus, MICB locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

In some embodiments, the cell is a T cell derived from an induced pluripotent stem cell comprising one or more of the following that reduces expression: endogenous T cell receptors; cytotoxic T-lymphocyte-associated protein 4 (CTLA 4); programmed cell death (PD 1); and programmed cell death ligand 1 (PD-L1), wherein the reduced expression is due to the modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification. In some embodiments, the cell is a T cell derived from an induced pluripotent stem cell comprising reduced expression of TRAC and TRB.

In some embodiments, the patient exhibits one or more of the following: (i) After administration of the population of cells, there is no systemic TH1 activation; (ii) Immune activation of Peripheral Blood Mononuclear Cells (PBMCs) is absent following administration of the population of cells; (iii) After administration of the population of cells, no donor-specific IgG antibodies are directed against the population of cells; (iv) Upon administration of the population of cells, no IgM and IgG antibodies are produced to the population of cells; and (v) no cytotoxic T cell killing of the population of cells after administration of the population of cells.

In some embodiments, the second administration is initiated when cells from the first administration are no longer detectable in the patient.

In some embodiments, the method further comprises administering the dosing regimen at least twice.

In one aspect, there is provided the use of a population of low immunity cells for treating a disorder in a patient, wherein the low immunity cells comprise a first exogenous polynucleotide encoding CD47, a second exogenous polynucleotide encoding a CAR, and (I) one or more of: (a) Major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression; (b) reduced expression of MHC class I and class II human leukocyte antigens; (c) Reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transactivator (CIITA); and/or (d) reduced expression of B2M and CIITA; wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification; (II) wherein: the patient is not a sensitive patient; or the patient is a sensitive patient.

In some embodiments, the patient is a sensitive patient, and wherein the patient exhibits a memory B-cell and/or memory T-cell response against one or more alloantigens or one or more autoantigens.

In some embodiments, the one or more alloantigens comprise human leukocyte antigens.

In some embodiments, the patient is a sensitive patient that is sensitive to the disorder or previous treatment of the disorder. In some embodiments, the patient receives a prior treatment for the disorder or disease, wherein the prior treatment does not comprise the population of cells, and wherein: (a) Administering the population of cells to treat the same disorder or disease previously treated; (b) The population of cells exhibits an enhanced therapeutic effect on the treatment of a disorder or disease in a patient compared to prior treatments; (c) The population of cells exhibits a longer therapeutic effect on the treatment of a disorder or disease in a patient than prior treatments; (d) the prior treatment is therapeutically effective; (e) prior treatment is treatment ineffective; (f) the patient develops an immune response to the previous treatment; and/or (g) administering the population of cells to treat a condition or disease that is different from the previous treatment. In some embodiments, the prior treatment comprises administering a population of therapeutic cells comprising a suicide gene or safety switching system, and an immune response occurs in response to activation of the suicide gene or safety switching system. In some embodiments, the prior treatment comprises a mechanically assisted treatment, optionally, wherein the mechanically assisted treatment comprises hemodialysis or ventricular assist devices.

In some embodiments, the cells differentiate from stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a pluripotent stem cell, optionally, wherein the pluripotent stem cell is an induced pluripotent stem cell. In some embodiments, the cell is a CAR T cell or CAR-NK cell. Cells differentiate from stem cells. In some embodiments, the cells are derived from primary T cells. In some embodiments, the cells are derived from a T cell pool comprising primary T cells from one or more subjects other than the patient.

In some embodiments, the antigen binding domain of the CAR binds to CD19, CD22, or BCMA. In some embodiments, the CAR is a CD 19-specific CAR, such that the cell is a CD19 CAR T cell. In some embodiments, the CAR is a CD 22-specific CAR, such that the cell is a CD22CAR T cell. In some embodiments, the cell comprises a CD 19-specific CAR and a CD 22-specific CAR, such that the cell is a CD19/CD22CAR T cell. In some embodiments, the CD 19-specific CAR and the CD 22-specific CAR are encoded by a single bicistronic polynucleotide. In some embodiments, the CD 19-specific CAR and the CD 22-specific CAR are encoded by two separate polynucleotides.

In some embodiments, the safe harbor locus is selected from the group consisting of: CCR5 gene locus, PPP1R12C (also known as AAVS 1) gene and CLYBL gene locus. In some embodiments, the target locus is selected from the group consisting of: CXCR4 locus, albumin locus, SHS231 locus, ROSA26 locus, CD142 locus, MICA locus, MICB locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus. In some embodiments, the insertion into the CCR5 gene locus is at exons 1 to 3, introns 1 to 2, or another coding sequence (CDS) of the CCR5 gene. In some embodiments, the insertion into the PPP1R12C gene locus is intron 1 or intron 2 of the PPP1R12C gene. In some embodiments, the insertion into the CLYBL gene locus is intron 2 of the CLYBL gene.

In some embodiments, the insertion into the ROSA26 gene locus is intron 1 of the ROSA26 gene. In some embodiments, the insertion into the safe harbor locus is the SHS231 locus. In some embodiments, the insertion into the CD142 gene locus is at exon 2 of the CD142 gene or another CDs. In some embodiments, the insertion into the MICA gene locus is at the CDS of the MICA gene. In some embodiments, the insertion into the MICB gene locus is at the CDS of the MICB gene. In some embodiments, the insertion into the B2M gene locus is at exon 2 of the B2M gene or another CDS. In some embodiments, the insertion into the CIITA gene locus is at exon 3 of the CIITA gene or another CDS. In some embodiments, the insertion into the TRAC gene locus is at exon 2 of the TRAC gene or another CDS. In some embodiments, the insertion into the TRB gene locus is at the CDS of the TRB gene.

In some embodiments, the primary T cell-derived cells comprise one or more of the following that reduce expression: (a) an endogenous T cell receptor; (b) Cytotoxic T-lymphocyte-associated protein 4 (CTLA 4); (c) programmed cell death (PD 1); and (d) a programmed cell death ligand 1 (PD-L1), wherein the reduced expression is due to the modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification.

In some embodiments, the cells derived from primary T cells comprise reduced expression TRAC.

In some embodiments, the patient exhibits no immune response after administration of the population of cells. In some embodiments, the immune-free response after administration of the population of cells is selected from the group consisting of: no systemic immune response, no adaptive immune response, no innate immune response, no T cell response, no B cell response, and no systemic acute cellular immune response. In some embodiments, the patient exhibits one or more of the following: (a) After administration of the population of cells, there is no systemic TH1 activation; (b) Immune activation of Peripheral Blood Mononuclear Cells (PBMCs) is absent following administration of the population of cells; (c) After administration of the population of cells, no donor-specific IgG antibodies are directed against the population of cells; (d) Upon administration of the population of cells, no IgM and IgG antibodies are produced to the population of cells; and (e) no cytotoxic T cell killing of the population of cells after administration of the population of cells.

In some embodiments, the method comprises a dosing regimen comprising: (a) A first administration comprising a therapeutically effective amount of the population of cells; (b) during recovery; and (c) a second administration comprising a therapeutically effective amount of the population of cells. In some embodiments, the recovery period comprises at least 1 month or more. In some embodiments, the recovery period comprises at least 2 months or more. In some embodiments, the second administration is initiated when cells from the first administration are no longer detectable in the patient. In some embodiments, the low immunity cells are removed by suicide genes or safety switching systems, and wherein the second administration is initiated when cells from the first administration are no longer detectable in the patient. In some embodiments, the use of the cells provided herein further comprises administering the dosing regimen at least twice.

In some embodiments of the use or method, the prior treatment comprises an allogeneic CAR-T cell-based therapy or an autologous CAR-T cell-based therapy, wherein the autologous CAR-T cell-based therapy is selected from the group consisting of: bucarba Ji Aolun, sicalico, ai Kaba Ji Weisai, li Jimai Racemosaicism, te Sha Jinlu, descartes-08 or Descartes-11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Cellectis, PBCAR19B or PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics and CYAD-211 from Clyad Oncology.

Drawings

FIGS. 1A through 1F are a representative set of ELISPOT quantification of serum from NHP cross-administered wild type human (FIGS. 1A, 1B, 1D and 1F) and HIP (FIGS. 1A, 1C, 1D and 1E) iPSCs. FIGS. 1A through 1C show the receipt of wild type human iPSC (wt) at the time of first injection ^xeno ) Weight was received at the second injection ^xeno And receiving a human HIPiPSC (HIP) at the time of the third injection ^xeno ) Is a study group result. FIGS. 1D through 1F show HIP receiving upon first injection ^xeno Receiving HIP at the second injection ^xeno And receiving wt at the time of the third injection ^xeno Is a study group result. At the receiving wt ^xeno Injection and HIP ^xeno All assay runs after injection are shown as horizontal bars and vertical bars, respectively. Blood drawing analysis is administered to cells at various time points, e.g., before treatment ("pre-Tx"), on day 7, day 13, day 75, and after, including on cross-injection ("pre-Tx") and on day 7, day 13, and day 75 thereafter. The symbols of days in parentheses below represent the time of blood drawing relative to the first injection (first column), second injection (second column) and third injection (third column), as shown in fig. 1A to 7C, 8C and 8E.

FIGS. 2A and 2B are a representative set of graphs showing donor-specific IgG antibody binding in serum of NHP cross-administered wild-type (FIG. 2A) or HIP (FIGS. 2A and 2B) human iPSC. FIGS. 2A and 2B show the receipt of wt at the time of first injection ^xeno Weight was received at the second injection ^xeno And receiving HIP at the time of the third injection ^xeno Is a study group result. In FIG. 2A, for wt ^xeno And HIP ^xeno Is shown as a circle with a horizontal line and a circle with a vertical line, respectively. FIG. 2B shows acceptance of HIP ^xeno IgG DSA amount after injection.

Fig. 3A and 3B are a representative set of graphs showing donor-specific IgG antibody binding in serum of NHP cross-administered wild-type (fig. 3A and 3B) or HIP (fig. 3A) human ipscs. FIGS. 3A and 3B show HIP reception at first injection ^xeno HIP was received at the time of the second injection ^xeno And receiving wt at the time of the third injection ^xeno Is a study group result. In FIG. 2A, for wt ^xeno And HIP ^xeno Is shown as a circle with a horizontal line and a circle with a vertical line, respectively. FIG. 3B shows the accepted wt ^xeno IgG DSA amount after injection.

Fig. 4A to 4C are a representative set of graphs showing total IgM antibodies in serum of NHP cross-administered wild-type (fig. 4A and 4B) or HIP (fig. 4A and 4C) human ipscs. FIGS. 4A to 4C show the following stepsReceiving human HIPiPSC (HIP) at one injection ^xeno ) HIP was received at the time of the second injection ^xeno And receiving wt at the time of the third injection ^xeno Is a study group result. FIG. 4B shows the accepted wt ^xeno Total IgM antibody amount after injection and figure 4C shows HIP received at the second injection ^xeno Total IgM antibody amount after.

Fig. 5A to 5C are a representative set of graphs showing total IgM antibodies in serum of NHP cross-administered wild-type (fig. 5A and 5B) or HIP (fig. 5A and 5C) human ipscs. FIGS. 5A to 5C show the receipt of wt at the time of first injection ^xeno Weight was received at the second injection ^xeno And receiving HIP at the time of the third injection ^xeno Is a study group result. FIG. 5B shows the wt was received at the time of the second injection ^xeno Post total IgM antibody levels, and figure 5C shows HIP exposure at the third injection ^xeno Total IgM antibody amount after.

Fig. 6A to 6C are a representative set of graphs showing total IgG antibodies in serum of NHP cross-administered wild-type (fig. 6A and 6B) or HIP (fig. 6A and 6C) human ipscs. FIGS. 6A through 6C show HIP reception at the time of first injection ^xeno HIP was received at the time of the second injection ^xeno And receiving wt at the time of the third injection ^xeno Is a study group result. FIG. 6B shows the wt was received at the third injection ^xeno The total IgG antibody amount after the second injection and figure 6C shows HIP ^xeno Total IgG antibody amount after.

Fig. 7A to 7C are a representative set of graphs showing total IgG antibodies in serum of NHP cross-administered wild-type (fig. 7A and 7B) or HIP (fig. 7A and 7C) human ipscs. FIGS. 7A through 7C show HIP reception at the time of first injection ^xeno Weight was received at the second injection ^xeno And receiving HIP at the time of the third injection ^xeno Is a study group result. FIG. 7B shows the wt was received at the time of the second injection ^xeno The total IgG antibody amounts after the third injection and figure 7C shows HIP ^xeno Total IgG antibody amount after.

Fig. 8A to 8E are a representative set of graphs showing that Natural Killer (NK) cell-mediated killing of HIP human ipscs differentiated into wild-type NHPs is absent. FIGS. 8A to 8C show the reception of HI at first injectionP ^xeno HIP was received at the time of the second injection ^xeno And receiving wt at the time of the third injection ^xeno NK cell mediated killing in the study group. NK cell-killing without human HIP iPSC at the first injection stage (fig. 8A) and the second injection stage (fig. 8B) is depicted in the instant cell biosensor data plot. FIGS. 8D and 8E show the wt received at the first injection ^xeno Weight was received at the second injection ^xeno And receiving HIP at the time of the third injection ^xeno NK cell mediated killing in the study group. NK cell-killing without human HIP iPSC at the third injection stage (fig. 8D) was plotted in the immediate cell biosensor data plot. Percent target cell killing is shown on the left y-axis (mean ± s.d.), killing rate on the right y-axis (killing t _1/2 ^-1 Mean ± s.e.m.; shown as a hollow triangle). At the receiving wt ^xeno And HIP ^xeno Post-injection assay runs are shown as circles with horizontal lines and circles with vertical lines, respectively.

Fig. 9A shows representative BLI images of transplanted HIP rhesus ipscs in the left leg of allogeneic NHP recipients. The BLI signal over time and percent BLI signal over time are shown in the BLI images in figures 9A, 10, 11, 12A through 12B and 13C below, relative to day 0 or pre-transplant amount. FIG. 9B is an immunohistological image of tissue from the injection site 6 weeks after implantation. The images show SMA positive blood vessels and luciferase positive cells, indicating transplanted HIP rhesus ipscs and their progeny.

Fig. 10 shows representative BLI images of transplanted wild-type rhesus ipscs (top column) of the left leg of an allogeneic NHP recipient and transplanted HIP rhesus ipscs (bottom column) of the right leg of the same recipient, which were sensitized 5 weeks after the wild-type rhesus iPSC graft.

Fig. 11 shows representative BLI images of transplanted wild-type rhesus ipscs (top column) on the left leg of another allogeneic NHP recipient and transplanted HIP rhesus ipscs (bottom column) on the right leg of the same recipient, which were sensitized 5 weeks after the wild-type rhesus iPSC grafts.

Figures 12A and 12B show representative BLI images of allogeneic NHP recipients from a cross-study of HIP rhesus ipscs to wild-type rhesus ipscs. The top row shows images of transplanted HIP rhesus ipscs and their offspring for the left leg of an allogeneic NHP recipient, while the bottom row shows transplanted wild-type rhesus ipscs for the right leg of the same recipient. The lower right hand corner also depicts images of HIP rhesus ipscs and their offspring transplanted in the left leg of allogeneic NHP recipients 8 weeks and 9 weeks after initial HIP iPSC transplantation.

Fig. 13A shows representative BLI signals over time for representative allogeneic NHP recipients initially transplanted with wild-type rhesus ipscs in their left legs and HIP rhesus ipscs in their right legs after injection. Figure 13B shows representative BLI signals over time for representative allogeneic NHP recipients initially transplanted with HIP rhesus ipscs in the left leg of the allogeneic NHP recipient and wild-type rhesus ipscs in the right leg of the same recipient after cross injection. Fig. 13C shows representative BLI images of allogeneic NHP recipients of the administered HIP rhesus ipscs injected into the left leg for the first time from day 0 to week 9.

Figures 14A to 14G show characterization of human wt and HIP ipscs prior to xenograft to NHP recipients. FIGS. 14A and 14B show wt ^xeno (FIG. 14A) and HIP ^xeno (FIG. 14B) morphology of the cultures. In wt ^xeno (FIG. 14C) and HIP ^xeno The surface expression of HLA class I and class II and CD47 on (fig. 14D) was assessed by flow cytometry and plotted as histograms. FIG. 14E shows wt before implantation ^xeno And HIP ^xeno Is a cell preparation of the cell preparation. The viability of NHP recipients was higher than 90% (mean ± s.d.). FIG. 14F shows subcutaneous injection of wt ^xeno Representative BLI images and BLI signals over time of NSG mice of iPSC. FIG. 14G shows subcutaneous injection HIP ^xeno Representative BLI images and BLI signals over time of NSG mice of iPSC.

Figures 15A to 15J show the characterization of rhesus wt and HIP ipscs prior to allogeneic transplantation into NHP recipients. FIGS. 15A to 15C show wt ^allo (FIG. 15A) and HIP ^allo (FIGS. 15B and 15C) morphology of the cultures. In wt ^allo (FIG. 15D) and HIP ^allo Surface expression of HLA class I and class II and CD47 on (FIGS. 15E and 15F)Assessed by flow cytometry and depicted as histograms. FIG. 15G shows wt before implantation ^allo HIP (high performance liquid chromatography) ^allo Is a cell preparation of the cell preparation. Viability into NHP recipients was higher than 90% (mean ± s.d.). FIG. 15H shows subcutaneous injection wt ^allo Representative BLI images and BLI signals over time of NSG mice of iPSC. FIGS. 15I and 15J show subcutaneous injection HIP ^allo Representative BLI images and BLI signals over time of NSG mice of iPSC.

FIG. 16 is an illustration of an evaluation of B2M ^indel/indel 、CIITA ^indel/indel Representative plots of CD47 expression in CD47tg iPSC. In these ipscs, the CD47 transgene was inserted into a safe harbor site (AAVS 1, CYBL, or CCR 5), and the CAG or EF1 alpha promoter was used to control expression of the CD47 polynucleotide. As shown in the figure, B2M ^indel/indel 、CIITA ^indel/indel CD47tg iPSC expressed CD47 about-30 to 200 fold higher than baseline.

FIG. 17 is an illustration of an evaluation of B2M ^indel/indel 、CIITA ^indel/indel Representative plots of CD47 expression in CD47tg iPSC. In these ipscs, the CD47 transgene was inserted into the CYBL safe harbor site, and the EF1 a promoter was used to control expression of the CD47 polynucleotide. As shown, in P23 and P27, B2M ^indel/indel 、CIITA ^indel/indel CD47tg iPSC over-expressed CD47.

FIG. 18 is a graph of the evaluation at B2M at various time points (P20, P21, P23 and P27) ^indel/indel 、CIITA ^indel/indel Representative plots of CD47 expression in CD47tg iPSC. In these ipscs, the CD47 transgene was inserted into CCR5 or CLYBL safe harbor sites, and the CAG or EF1 a promoters were used to control expression of the CD47 polynucleotide. As shown, at different time points, B2M ^indel/indel 、CIITA ^indel/indel CD47tg iPSC over-expressed CD47.

FIGS. 19A to 19C are diagrams for evaluation of B2M by innate immune cells (NK cells and macrophages) ^indel/indel 、CIITA ^indel/indel Representative graphs of studies of killing of CD47tg iPSC. B2M ^indel/indel And CIITA ^indel/indel The CD47tg of iPSC was inserted into the safe harbor site (AAVS 1, CYBL or CCR 5). As shown, all cellsThe clones were protected from NK and macrophage cell killing.

Other objects, advantages and embodiments of the present technology will be apparent from the detailed description that follows.

Detailed Description

I. Introduction to the invention

The present disclosure relates to methods and compositions for reducing and/or avoiding the effects of immune system responses on cell therapies. To overcome the problem of immune rejection in subjects of cell derived and/or tissue grafts, the inventors herein developed and disclosed immune evasive (e.g., low immune cells or low immune multipotent cells) cells that represent a viable source of any transplantable cell type. Advantageously, the cells disclosed herein are not rejected by the immune system of the recipient subject, regardless of the subject's genetic make-up (or any existing response in the subject to one or more prior allogeneic or autologous cell-derived and/or tissue grafts.

The technology disclosed herein utilizes genetic modification to modulate (e.g., reduce or eliminate) MHC I and/or MHC II expression. In some embodiments, genome editing techniques that utilize rare-cutting (rare-cutting) endonucleases (e.g., CRISPR/Cas, TALENs, zinc finger nucleases, meganuclease (meganuclease) and homing (home) endonuclease systems) are also used to reduce or eliminate gene expression in human cells that involves an immune response (e.g., by deleting genomic DNA of genes involved in an immune response, or by inserting genomic DNA into such genes such that gene expression is affected). In certain embodiments, genome editing techniques or other gene modulation techniques are used to insert tolerance-inducing (tolerogenic) factors in human cells so that they, and differentiated cells prepared from such cells, can escape immune recognition when implanted into a recipient subject. Thus, the cells described herein exhibit modulated expression of one or more genes and/or factors that affect MHC I and/or MHC II expression.

The genome editing techniques described herein are capable of double-stranded DNA breaks at desired locus sites. These controlled double strand breaks promote homologous recombination at specific locus sites. This process focuses on targeting specific sequences of nucleic acid molecules (such as chromosomes) with endonucleases that recognize and bind to the sequences and induce double strand breaks in the nucleic acid molecules. Double strand breaks are repaired by error-prone non-homologous end joining (NHEJ) or Homologous Recombination (HR).

Certain genome editing techniques described herein are capable of single-stranded DNA breaks at desired locus sites, wherein base editing or primary editing can be used to sequentially change a single nucleobase to a surrogate base to change the genomic sequence. In some embodiments, base editing is used to modulate MHC I and/or MHC II antigens, tolerogenic factors, and/or CAR expression. Description of base editing can be found, for example, in Rothgangl et al, nat Biotechnol,2021,39,949-957; porto et al, nat Rev Drug Discov,2020,19,839-859; and Rees and Lui, nat Rev Genet,2018,19 (12), 770-788. In some embodiments, the primary editing is used to modulate MHC I/or MHC II antigen tolerogenic factors and/or CAR expression. Descriptions of the primary edits may be found, for example, in Anzalone et al, nature,2019,576,149-157; kantor et al, int J mobile Sci,2020,21 (17), 6240; schene et al, nat Commun,2020,11,5232; and Scholef field and Harrison, gene Therapy,2021, doi.org/10.1038/s 41434-021-00263-9.

Unless specifically indicated as antisense, the practice of particular embodiments will employ routine methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology within the skill of the art, many of which are described below for purposes of illustration. These techniques are well explained in the literature. See, for example, sambrook et al Molecular Cloning: A Laboratory Manual (3 rd edition, 2001); sambrook et al Molecular Cloning: A Laboratory Manual (2 nd edition, 1989); maniatis et al, molecular Cloning: A Laboratory Manual (1982); ausubel et al Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology Greene Pub.associates and Wiley-Interscience; glover, DNA Cloning: A Practical Approach, vol.I & II (IRL Press, oxford, 1985); anand, techniques for the Analysis of Complex Genomes, (Academic Press, new York, 1992); transcription and Translation (b.hames & s.higgins, eds., 1984); perbal, A Practical Guide to Molecular Cloning (1984); harlow and Lane, antibodies, (Cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q.E.Coligan, A.M.Kruisbeek, D.H.Margulies, E.M.Shevach and W.Strober, eds., 1991); annual Review of Immunology; and monographs on journals, such as Advances in Immunology.

II. Definition of

The term "autoimmune disease" refers to any disease or disorder in which a subject initiates a destructive immune response to self-tissues and/or cells. Autoimmune disorders can affect almost every organ system of a subject (e.g., a human), including, but not limited to, diseases of the nerve, gastrointestinal and endocrine systems, as well as skin and other connective tissue, eyes, blood and blood vessels. Examples of autoimmune diseases include, but are not limited to, hashimoto's thyroiditis, systemic lupus erythematosus, sjogren's syndrome, grave's disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

The term "cancer" as used herein is defined as the hyper-proliferation of cells whose unique characteristics (e.g., loss of normal control) result in unregulated growth, lack of differentiation, localized tissue invasion and metastasis. With respect to the methods of the invention, the cancer may be any cancer, including any acute lymphocytic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal canal cancer or anorectal (anoectoum) cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulval cancer, chronic lymphocytic leukemia, chronic myelogenous cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer, lymphoma, malignant mesothelioma, obesity cell tumor, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, cancer, testicular cancer, bladder cancer, ureteral cancer, urinary bladder cancer or urinary bladder cancer. As used herein, unless specifically indicated otherwise, the term "tumor" refers to abnormal growth of malignant type cells or tissue, and excludes benign type tissue.

The term "chronic infectious disease" refers to a disease caused by an infectious agent, wherein infection is persistent. Such diseases may include hepatitis (A, B or C), herpes viruses (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV and EBV), and HIV/AIDS. Non-viral examples may include chronic fungal diseases such as aspergillosis, candidiasis, globosum and cryptococcus-related diseases and histoplasmosis. Non-limiting examples of chronic bacterial infectious pathogens may be C.pneumoniae, L.monocytogenes and M.tuberculosis. In some embodiments, the disorder is Human Immunodeficiency Virus (HIV) infection. In some embodiments, the disorder is acquired immunodeficiency syndrome (AIDS).

In some embodiments, the alterations or modifications described herein (including, e.g., genetic alterations or modifications) result in a target or selected polynucleotide sequence that reduces expression. In some embodiments, the changes or modifications described herein result in a target or selected polypeptide sequence that reduces expression. In some embodiments, the changes or modifications described herein result in a target or selected polynucleotide sequence that increases expression. In some embodiments, the alterations or modifications described herein result in increased expression of the target or selected polypeptide sequence. The terms "reduce", "decrease" and "reduction" are used herein to mean generally a statistically significant amount of reduction. However, for the avoidance of doubt, "reduced" and "reduced" mean at least a 10% reduction compared to a reference level, for example at least about 20% reduction, 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 and including a 100% reduction (i.e. no level compared to a reference sample) or a 10 to 100% reduction compared to a reference level. In some embodiments, the cells are engineered to have one or more targets of reduced expression relative to an unaltered or unmodified wild type cell. In the context of cells, "wild-type" or "wt" refers to any cell found in nature. However, for example, in the context of an engineered cell or a low immunity cell, as used herein, "wild-type" may also refer to a nucleic acid alteration that may comprise MHC I and/or II and/or T-cell receptors that result in reduced expression, but not subjected to a gene editing program to result in over-expression of CD47 protein, e.g., the cell may be a "wild-type" of CD47, but altered in relation to MHC I and/or II and/or T-cell receptors. As used herein, "wild-type" may also refer to an engineered cell or a low immunity cell that may contain an alteration in a nucleic acid that results in overexpression of the CD47 protein, but not subjected to a gene editing program to result in reduced expression of MHC I and/or II and/or T-cell receptors, e.g., the cell may be "wild-type" for MHC I and/or II and/or T-cell receptors, but altered with respect to CD 47. In the context of PSCs or their progeny, "wild-type" also refers to PSCs or their progeny that may contain nucleic acid alterations that result in pluripotency, but have not undergone the genetic editing procedures of the present technology to achieve overexpression of reduced expression of MHC I and/or II and/or T-cell receptor and/or CD47 proteins. Also in the context of PSCs or their progeny, "wild-type" refers to PSCs or their progeny that may contain nucleic acid alterations that result in overexpression of CD47 protein, but have not undergone a gene editing program to result in reduced expression of MHC I and/or II and/or T-cell receptors. In the context of primary cells or their progeny, "wild-type" also refers to primary cells or their progeny that may contain nucleic acid alterations that reduce the expressed MHC I and/or II and/or T-cell receptors, but have not undergone a gene editing program to result in overexpression of the CD47 protein. Also in the context of primary cells or their progeny, "wild-type" refers to primary cells or their progeny that may contain a nucleic acid alteration that results in overexpression of the CD47 protein, but have not undergone a gene editing program to result in reduced expression of MHC I and/or II and/or T-cell receptors. In some specific embodiments, the cells are engineered to have one or more targets of reduced or increased expression relative to cells of the same cell type that do not comprise the modification.

The term "endogenous" refers to a reference molecule or polypeptide that naturally occurs in a cell. Likewise, when used in reference to expression of a coding nucleic acid, the term refers to expression of the coding nucleic acid that is naturally contained within a cell rather than being exogenously introduced.

As used herein, the term "exogenous" is intended to mean that the molecule of interest or the polypeptide of interest is introduced into a cell of interest. For example, a polypeptide may be introduced by introducing a nucleic acid encoding a gene material into a cell, such as by integration into a chromosome or as a non-chromosomal gene material, such as a plasmid or expression vector. Thus, when used in reference to expression of a coding nucleic acid, the term refers to the introduction of the coding nucleic acid into a cell in an expressible form. An "exogenous" molecule is a molecule, construct, factor, etc. that is not normally present in a cell, but can be introduced into the cell by one or more genes, biochemistry or other means. The "normal presence in a cell" is determined by the specific developmental stage and environmental conditions associated with the cell. Thus, for example, a molecule that is present only during neuronal embryo development is an exogenous molecule relative to an adult neuronal cell. Exogenous molecules may include, for example, a functional version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functional endogenous molecule.

The exogenous molecule or factor may be, among other things, a small molecule, such as produced by a combinatorial chemical process or a large molecule, such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the foregoing, or a complex comprising one or more of the foregoing. Nucleic acids include DNA and RNA, which may be single-stranded or double-stranded; may be linear, branched or cyclic; and may be of any length. Nucleic acids include those capable of forming double strands, as well as triplex forming nucleic acids. See, for example, U.S. Pat. nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and/or helicases.

For the purposes of this disclosure, a "gene" includes a DNA region encoding a gene product, as well as all DNA regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to the coding and/or transcribed sequences. Thus, genes include, but are not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, border elements, origins of replication, matrix attachment sites, and/or locus control regions.

"Gene expression" refers to the conversion of information contained in a gene into a gene product. The gene product may be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribonuclease, structured RNA, or any other type of RNA) or a protein produced by mRNA translation. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation and editing and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation and/or glycosylation.

As used herein, the term "genetic modification" and grammatical equivalents thereof may refer to one or more alterations of a nucleic acid (e.g., a nucleic acid within the genome of an organism). For example, a genetic modification may refer to a change, addition, and/or deletion of a gene or portion of a gene or other nucleic acid sequence. Genetically modified cells may also refer to cells having added, deleted and/or altered genes or gene portions. Genetically modified cells may also refer to cells having added nucleic acid sequences that are not genes or portions of genes. Genetic modifications include, for example, both transient knock-in (knock-in) or knock-down (knock-down) mechanisms, and mechanisms that result in permanent knock-in, knock-out, or knock-out of a target gene or portion of a gene or nucleic acid sequence. Genetic modifications include, for example, knockins and mechanisms that result in permanent knockins of nucleic acid sequences.

As used herein, the terms "grafting", "administering", "introducing", "implanting" and "transplanting" and grammatical equivalents thereof are used interchangeably in the context of placement of cells (e.g., cells as described herein) into a subject, by a method or route that results in localized or at least partial localization of introduced cells at a desired site or systemic introduction (e.g., into the circulation). The cells may be implanted directly into the desired site or administered by any suitable route that results in delivery to the desired location within the subject, wherein at least a portion of the implanted cells or components of the cells remain viable. The viability period of the cells after administration to a subject may be as short as several hours, e.g., 24 hours, to several days, to as long as several years. In some embodiments, the cells may also be administered (e.g., injected) at a location other than the desired site, such as in the brain or subcutaneously, e.g., in a capsule, to maintain the implanted cells in the implantation site and avoid migration of the implanted cells.

An "HLA" or "human leukocyte antigen" complex refers to a complex of genes encoding human Major Histocompatibility Complex (MHC) proteins. These cell surface proteins constituting the HLA complex are responsible for the regulation of the immune response to the antigen. In humans, there are two classes of MHC, class I and class II, "HLA-I" and "HLA-II". HLA-I includes three proteins HLA-A, HLA-B and HLA-C, which present peptides from the cell interior, while antigens presented by HLA-I complexes attract killer T-cells (also known as CD8+ T-cells or cytotoxic T-cells). HLA-I cells are associated with beta-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens from outside the cell to T lymphocytes. This stimulates cd4+ cells (also known as T-helper cells). It will be appreciated that the use of "MHC" or "HLA" is not meant to be limiting, as this depends on whether the gene is from a Human (HLA) or Murine (MHC). Thus, these terms may be used interchangeably herein when referring to mammalian cells.

As used herein to characterize a cell, the term "hypoimmunity" generally refers to such a cell being less susceptible to immune rejection, e.g., congenital or adaptive immune rejection of such a cell transplanted into a subject, e.g., the cell is less susceptible to allogeneic rejection (alloreject) of such a cell transplanted into a subject. For example, such low immunity cells may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to immune rejection relative to unmodified or wild-type or non-low immunity cells, and thus the cells are transplanted into a subject. In some embodiments, genome editing techniques are used to modulate the expression of MHC I and MHC II genes, thereby facilitating the generation of low immunity cells. In some embodiments, the low-immunity cells escape immune rejection in MHC-mismatched allogeneic recipients. In some cases, differentiated cells produced by the low immunity stem cells outlined herein escape immune rejection when administered (e.g., transplantation (transplant) or grafting (graft)) to an MHC-mismatched allogeneic recipient. In some embodiments, the low immunity cells are protected from T cell mediated adaptive immune rejection and/or innate immune cell rejection. Detailed description of low immunity cells, methods of producing the same, and methods of using the same WO2016183041, filed 5, 9, 2015; WO2018132783 filed on 14 days 1 month 2018; WO2018176390 filed on 20/3/2018; WO2020018615 filed on 7 months 17 2019; WO2020018620 filed on 7 months 17 2019; PCT/US2020/44635 filed on 31 th 7 th 2020; US62/881,840 filed on 1/8/2019; US62/891,180 filed on 8 months 23 in 2019; US63/016,190 of the application of month 27 of 2020; and U.S. patent No. 63/052,360 to 7/15 of 2020, the disclosures of which including the embodiments, sequence listing and drawings are incorporated herein by reference in their entirety.

The hypoimmunity of a cell can be determined by assessing the immunity of the cell, e.g., the ability of the cell to elicit or avoid eliciting an adaptive and innate immune response. This immune response may be measured using assays recognized by those of ordinary skill in the art to which the invention pertains. In some embodiments, the immune response assay measures the effect of low immune cells on T cell proliferation, T cell activation, T cell killing, donor specific antibody production, NK cell proliferation, NK cell activation, and macrophage activity. In some examples, the low immunity cells and derivatives thereof undergo killing by T-cell and/or NK cell depletion upon administration to a subject. In some examples, the cells and derivatives thereof exhibit reduced phagocytosis by macrophages compared to unmodified or wild-type cells. In some embodiments, the low-immunity cell is non-immune or incapable of eliciting an immune response in the recipient subject.

The term percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of identical nucleotide or amino acid residues when compared and aligned for maximum identity, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to a person of ordinary skill), or by visual inspection. Depending on the application, a percentage "identity" may be present in the region of the sequences being compared, for example, in the functional domain, or over the full length of the two sequences to be compared. For sequence comparison, a sequence is typically used as a reference sequence for comparison with the test sequence. When using the sequence comparison algorithm, the test sequence and the reference sequence are entered into a computer, the subsequence coordinates are designated, and the sequence algorithm program parameters are designated, if necessary. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

Optimal alignment of sequences for comparison can be performed, for example, by Smith & Waterman, adv.appl.math.2:482 The local homology algorithm of (1981) was performed by Needleman & Wunsch, j.mol. Biol.48:443 The homology alignment algorithm of (1970) was performed by Pearson & Lipman, proc.Natl. Acad.Sci.USA 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, genetics Computer Group,575Science Dr., madison, wis.) or by visual inspection (see Ausubel et al in general, see below).

One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J.mol. Biol.215:403-410 (1990). Software for performing BLAST analysis is publicly available through National Center for Biotechnology Information.

As used herein, "immune signaling factor" refers in some examples to molecules, proteins, peptides, etc. that activate the immune signaling pathway.

As used herein, "immunosuppressive factor" or "immunomodulatory factor" or "tolerogenic factor" includes hyperimmune factors, complement inhibitors, and other factors that modulate or affect the ability of cells to be recognized by the immune system of a host or recipient subject after administration, transplantation, or implantation. These may be combined with additional genetic modifications.

The terms "increased", "increase" or "enhancement" or "activation" are used herein to refer generally to a statically significant amount of increase; for the avoidance of any doubt, the term "increased", "increased" or "enhanced" or "activated" refers to an increase of at least 10% compared to a reference level, for example 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 and including any increase of 100% or between 10 and 100% or at least about 2 times or at least about 3 times or at least about 4 times or at least about 5 times or at least about 10 times or any increase or higher than a reference level.

In some embodiments, the change is to indels. As used herein, "indel" refers to a mutation resulting from an insertion, a deletion, or a combination thereof. It will be appreciated by those of ordinary skill in the art that indels in the coding region of a genomic sequence will result in frame shift mutations unless the length of the indels is a multiple of three. In some embodiments, the alteration is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one of the nucleotides. The CRISPR/Cas systems of the present disclosure can be used to induce indels or point mutations of any length in a polynucleotide sequence of interest, for example using gene editing, base editing, or primary editing. The term "base editing" refers to a method of programmably converting one base pair to another at a targeted gene locus, and in some examples, does not make double-stranded DNA breaks, and in other cases, does not make single-stranded DNA breaks. In some embodiments, base editing utilizes catalytically compromised Cas9 to recognize the target DNA site, and has a range of PAM sequence recognition, a basal editing window within and/or outside the original spacer (protospacer) sequence. The term "primary editing" refers to a method of gene editing using a programmable polymerase (such as, but not limited to, napDNAbps as described in WO 2020191242) and a specific guide RNA. In some embodiments, the guide RNA includes a DNA synthesis template for encoding (or for deleting) gene information, which template is incorporated into the target DNA sequence. As recognized by one of ordinary skill in the art, base editing and primary editing have utility for modulating (e.g., reducing, eliminating, increasing, and enhancing) the expression of the described polynucleotides and polypeptides.

As used herein, "knock out" and "attenuation" refer to genetic modifications of an edited gene that result in no expression and reduced expression, respectively. As used herein, "attenuating" refers to a decrease in expression of a target mRNA or corresponding target protein. Attenuation is generally reported as the level present after administration or expression of a control molecule that does not mediate a reduced level of expression of RNA (e.g., non-targeted control shRNA, siRNA, guide RNA, or miRNA). In some embodiments, attenuating the target gene is achieved by shRNA, siRNA, miRNA or CRISPR interference (CRISPRi). In some embodiments, attenuating the target gene is achieved by a protein-based method, such as a degradation determinant method. In some embodiments, attenuating the target gene is achieved by genetic modification, including shRNA, siRNA, miRNA or using a gene editing system (e.g., CRISPR/Cas).

Attenuation is typically assessed by measuring mRNA levels using quantitative polymerase chain reaction (qPCR) amplification or protein levels by western blot methods or enzyme-linked immunosorbent assays (ELISA). Analysis of protein levels provides an assessment of mRNA cleavage and translational inhibition. Other techniques for measuring attenuation include RNA solution hybridization, nuclease protection, northern hybridization, gene expression monitoring using microarrays, antibody binding, radioimmunoassay, and fluorescence-activated cell analysis. One of ordinary skill in the art of the invention will readily understand how to use the gene editing system (e.g., CRISPR/Cas) of the present disclosure to knockout a polynucleotide sequence of interest, or a portion thereof, based on the details described herein.

"knock-in" herein refers to a genetic modification resulting from insertion of a DNA sequence into a chromosomal locus of a host cell. This results in increased expression levels of the knocked-in gene, portion of the gene, or product of nucleic acid sequence insertion, e.g., increased levels of RNA transcription and/or encoded protein. As will be appreciated by those of ordinary skill in the art, this can be accomplished in a variety of ways, including inserting or adding one or more additional copies of a gene or portion thereof into a host cell or altering regulatory components of an endogenous gene, to accomplish a specific nucleic acid sequence that increases protein expression or inserts the desired expression. This can be achieved by modifying the promoter, adding different promoters, adding enhancers, adding other regulatory elements or modifying other gene expression sequences. The CRISPR/Cas system of the present disclosure can be used to knock-in sequences, whether by homologous DNA repair using templates with homology arms or primary editing or gene writing, where specific sequences are edited. In some examples, the term "knock-in" refers to a process of adding a genetic function to a host cell. This results in increased levels of knockin gene products (e.g., RNA or encoded protein). As will be appreciated by those of ordinary skill in the art, this can be accomplished in a variety of ways, including adding one or more additional copies of a gene to a host cell or altering regulatory components of an endogenous gene, to accomplish increased protein expression. This can be achieved by modifying the promoter, adding a different promoter, adding an enhancer or modifying other gene expression sequences.

As used herein, "knockdown" includes deleting all or part of a target polynucleotide sequence in a manner that interferes with translation or function of the target polynucleotide sequence. For example, knock-out may be accomplished by altering a target polynucleotide sequence by inducing an insertion or deletion ("indel") in the target polynucleotide sequence at the target polynucleotide sequence, including a functional domain (e.g., a DNA binding domain) of the target polynucleotide sequence. One of ordinary skill in the art of the invention will readily understand how to use the gene editing system (e.g., CRISPR/Cas) of the present disclosure to knockout a polynucleotide sequence of interest, or a portion thereof, based on the details described herein.

In some embodiments, the genetic modification or change results in the knockdown or attenuation of the polynucleotide sequence of interest or a portion thereof. Gene editing systems (e.g., CRISPR/Cas) using the present technology to knockout a polynucleotide sequence of interest or portions thereof can be used in a variety of applications. For example, the knockdown of a polynucleotide sequence of interest in a cell may be performed in vitro for research purposes. For ex vivo purposes, the polynucleotide sequence of interest in the knockdown cell can be used to treat or prevent a disorder associated with expression of the polynucleotide sequence of interest (e.g., by knockdown of a mutant dual gene in an ex vivo cell and introducing those cells comprising the knockdown mutant dual gene into a subject) or to alter the genotype or phenotype of the cell. In some examples and as used herein, "knockout" includes deleting all or part of the polynucleotide sequence of interest in a manner that interferes with the function of the polynucleotide sequence of interest. For example, knock-out may be achieved by inducing an indel in the target polynucleotide sequence in a functional domain (e.g., a DNA binding domain) of the target polynucleotide to alter the target polynucleotide sequence. One of ordinary skill in the art of the invention will readily understand how to use the gene editing systems (e.g., CRISPR/Cas systems) of the present disclosure to knockout a polynucleotide sequence of interest, or a portion thereof, based on the details described herein. In some embodiments, the alteration results in the knockdown of the polynucleotide sequence of interest or a portion thereof. The use of CRISPR/Cas systems of the present disclosure to knock out a target polynucleotide sequence or portion thereof can be used in a variety of applications. For example, the knockdown of a polynucleotide sequence of interest in a cell may be performed in vitro for research purposes. For ex vivo purposes, the target polynucleotide sequence in the knockdown cell can be used to treat or prevent a disorder associated with expression of the target polynucleotide sequence (e.g., by knockdown of a mutant dual gene in the ex vivo cell and introducing those cells comprising the knockdown mutant dual gene into the subject).

"modulation" of gene expression refers to a change in the expression level of a gene. Modulation of expression may include, but is not limited to, gene activation and gene suppression. Modulation may also be complete, i.e., wherein gene expression is completely unactivated or activated to wild-type levels or exceeded; or it may be a part in which gene expression is partially reduced or partially activated to a part of the wild-type level.

In additional or alternative aspects, the present technology contemplates altering the target polynucleotide sequence in any manner available to a person of ordinary skill, for example, using a nuclease system, such as a TAL effector nuclease (TALEN) or Zinc Finger Nuclease (ZFN) system. It should be appreciated that although examples of methods utilizing CRISPR/Cas (e.g., cas9 and Cpf 1) with TALENs are described in detail herein, the techniques are not limited to use with these methods/systems. Other methods of targeting the reduction or elimination of expression in a target cell known to those of ordinary skill in the art of the invention may be utilized herein. The methods provided herein can be used to alter a polynucleotide sequence of interest in a cell. The present technology contemplates altering the target polynucleotide sequence in a cell for any purpose. In some embodiments, the polynucleotide sequence of interest in the cell is altered to produce a mutant cell. As used herein, "mutant cell" refers to a cell having a resulting genotype that is different from its original genotype. In some examples, a "mutant cell" exhibits a mutant phenotype, e.g., when a gene that is altered for normal function using the gene editing system of the present disclosure (e.g., CRISPR/Cas). In other examples, a "mutant cell" exhibits a wild-type phenotype, such as when the gene editing system (e.g., CRISPR/Cas) of the present disclosure is used to correct a mutant genotype. In some embodiments, the polynucleotide sequence of interest in the cell is altered to correct or repair the gene mutation (e.g., restore the normal phenotype of the cell). In some embodiments, the polynucleotide sequence of interest in the cell is altered to induce a mutation in the gene (e.g., to disrupt the function of the gene or genomic element).

The terms "operably connected," or "operably connected," are used interchangeably to refer to a juxtaposition of two or more components (e.g., sequential elements) wherein the components are configured such that the two components function properly and allow the possibility that at least one of the components may mediate a function imposed on at least one of the other components. For example, a transcriptional regulatory sequence (such as a promoter) is operably linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. The transcriptional regulatory sequence is typically, but not necessarily, operably linked in cis to the coding sequence. For example, enhancers are transcriptional regulatory sequences operably linked to a coding sequence even though they are not contiguous.

As used herein, a "pluripotent stem cell" has the potential to differentiate into any of three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, genitourinary tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue). The term "pluripotent stem cell" as used herein also encompasses "induced pluripotent stem cell" or "iPSC" or pluripotent stem cell types derived from non-pluripotent cells. In some embodiments, the pluripotent stem cells are produced or generated from cells that are not pluripotent cells. In other words, pluripotent stem cells may be the direct or indirect progeny of non-pluripotent cells. Examples of blast cells include somatic cells (reprogrammed by various means to induce a pluripotent, undifferentiated phenotype). Such "iPS" or "iPSC" cells may be created by inducing the expression of certain regulatory genes or by exogenous application of certain proteins. Methods of inducing iPS cells are known in the art and will be described further below. (see, e.g., zhou et al, stem Cells 27 (11): 2667-74 (2009), huangfu et al, nature Biotechnol.26 (7): 795 (2008), woltjen et al, nature 458 (7239): 766-770 (2009), and Zhou et al, cell Stem Cells 8:381-384 (2009), each of which is incorporated herein by reference in its entirety). The generation of Induced Pluripotent Stem Cells (iPSCs) is summarized below. As used herein, "hipscs" are human induced pluripotent stem cells.

"safe harbor locus" as used herein refers to a locus of genes that allows for a transgene or exogenous gene to be expressed in a manner that enables the predictable action of the newly inserted genetic element, and may also not cause alterations in the host genome in a manner that poses a risk to the host cell. Exemplary "safe harbor" loci include, but are not limited to, CCR5 genes, PPP1R12C (also known as AAVS 1) genes, CLYBL genes, and/or Rosa genes (e.g., rosa 26). "target locus" as used herein refers to a genetic locus that allows expression of a transgene or exogenous gene. Exemplary "target loci" include, but are not limited to, CXCR4 genes, albumin genes, SHS231 loci, F3 genes (also known as CD 142), MICA genes, MICB genes, LRP1 genes (also known as CD 91), HMGB1 genes, ABO genes, RHD genes, FUT1 genes, and/or KDM5D genes (also known as HY). Exogenous genes can be inserted into the CDs region of B2M, CIITA, TRAC, TRBC, CCR, F3 (i.e., CD 142), MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D (i.e., HY), PDGFRa, OLIG2, and/or GFAP. Exogenous genes can be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS 1) or CCR 5. Exogenous genes may be inserted into exons 1 or 2 or 3 of CCR 5. Exogenous genes can be inserted into intron 2 of CLYBL. Exogenous genes can be inserted into Ch-4:58,976,613 (i.e., SHS 231). The exogenous gene may be inserted into any suitable region of the safe harbor or target locus described above that allows expression of the exogenous gene, including, for example, an intron, exon, or coding sequence region in the safe harbor or target locus.

The terms "subject" and "individual" are used interchangeably herein and refer to an animal, e.g., a human, that provides a prophylactic treatment that can obtain cells therefrom and/or treat them, including using the cells described herein. For the treatment of an infection, disorder or disease state that is specific to a particular animal (such as a human subject), the term subject refers to a particular animal. "non-human animal" and "non-human mammal" as used interchangeably herein include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and/or non-human primates. The term "subject" also encompasses any vertebrate, including but not limited to mammals, reptiles, amphibians, and/or fish. Advantageously, however, the subject is a mammal, such as a human or other mammal, such as a domesticated mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc.

As used herein, the terms "treating" and "treatment" include administering to a subject an effective amount of a cell described herein such that the subject has a reduction in at least one symptom of a disease or an improvement in a disease, e.g., a beneficial or desired clinical outcome. Beneficial or desired clinical results in the art include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment may refer to prolonged survival compared to the expected survival without treatment. Thus, those of ordinary skill in the art of the invention understand that treatment may improve a disease condition, but may not cure the disease completely. In some embodiments, one or more symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the disease.

Beneficial or desired clinical results of a disease treatment in the context of the present technology include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

A "vector" or "construct" is capable of transferring a gene sequence to a cell of interest. Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing expression of a gene of interest and which can transfer a gene sequence to a cell of interest. Thus, the term includes cloning, and expression vectors (expression vehicle), as well as integration vectors. Methods for introducing vectors or constructs into cells are known to those of ordinary skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., micro-liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and/or viral vector-mediated transfer.

It should be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of such special purpose terms as "unique," "only," and the like in connection with the recitation of claim elements or use of a "negative" limitation. It will be apparent to those having ordinary skill in the art having access to the present disclosure that each of the individual embodiments described and illustrated herein has separate components and features that are readily separated from or combined with the features of any of the other several embodiments without departing from the scope and spirit of the present technology. Any recited method may be performed in the order of referenced events or any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the technology, the representative illustrative methods and materials are now described.

Before the technology is further described, it is to be understood that this technology is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present technology will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the technology. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the technology, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the technology. Certain ranges of values presented herein are preceded by the term "about. The term "about" is used herein to provide literal support for the exact number preceding it, as well as numbers near or approximating the term preceding. In determining whether a number is close or approximate to a specifically recited number, the close or approximate non-recited number may be a number that, in the context of presentation, provides a substantial equivalent of the specifically recited number.

All publications, patents, and patent applications cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Further, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter associated with the cited publication. Citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the art is not entitled to antedate such publication by virtue of prior art. Furthermore, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

Before the technology is further described, it is to be understood that this technology is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present technology will be limited only by the appended claims. It should also be understood that the headings used herein are not limiting and are intended only to guide the reader, but that the subject matter generally applies to the techniques disclosed herein.

Detailed description of the embodiments

A. Administering low immunity cells to a patient

In one aspect, provided herein is a method of treating a patient by administering a population of low immunity cells as described herein. The subject low immunity cells provided herein (e.g., cells differentiated from low immunity stem cells as described herein) can be administered to any suitable patient, including, for example, candidates for cell therapies for treating a disease or disorder. Candidates for cell therapy include any patient suffering from a disorder or disease that may benefit from the therapeutic effects of the subject hypoimmune cells provided herein. In some embodiments, the patient has a cellular defect. Candidates who may benefit from the therapeutic effects of the subject low immunity cells provided herein exhibit elimination, reduction, or amelioration of the disorder. As used herein, "cell defect" refers to any condition or disease that causes dysfunction or loss of a population of cells in a patient, wherein the patient is unable to naturally replace or regenerate the population of cells. Exemplary cellular defects include, but are not limited to, autoimmune diseases (e.g., multiple sclerosis, myasthenia gravis, rheumatoid arthritis, diabetes, systemic lupus erythematosus), neurodegenerative diseases (e.g., huntington's chorea and parkinson's disease), cardiovascular disorders and diseases, vascular disorders and diseases, corneal disorders and diseases, liver disorders and diseases, thyroid disorders and diseases, and/or kidney disorders and diseases. In some embodiments, the patient to whom the low immunity cells are administered has cancer. Exemplary cancers that can be treated by the low immunity cells provided herein include, but are not limited to, B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neublastic tumor, lung squamous cell carcinoma, hepatocellular carcinoma, and/or bladder cancer. In certain embodiments, a cancer patient is treated by administering a low immunity CAR-T-cell provided herein.

In some embodiments, the low immunity cells provided herein are useful for treating patients that are susceptible to one or more antigens present on a previous graft (such as, for example, cell grafts, blood transfusions, tissue grafts, and/or organ grafts). In certain embodiments, the prior graft is an allograft and the patient is sensitive to one or more alloantigens from the allograft. Allografts include, but are not limited to, allograft cell grafts, allograft blood transfusion, allograft tissue grafts and/or allograft organ grafts. In some embodiments, the patient is a susceptible patient who is pregnant or once pregnant (e.g., has or has had an alloimmune effect during pregnancy). In certain embodiments, the patient is sensitive to one or more antigens included in a previous graft, wherein the previous graft is a modified human cell, tissue, and/or organ. In some embodiments, the modified human cells, tissues and/or organs are modified autologous human cells, tissues and/or organs. In some embodiments, the previous graft is a non-human cell, tissue, and/or organ. In an exemplary embodiment, the previous graft is a modified non-human cell, tissue and/or organ. In certain embodiments, the previous graft is a chimera comprising a human component. In certain embodiments, the prior graft is and/or comprises CAR-T-cells. In certain embodiments, the previous graft is an autograft and the patient is sensitive to one or more autoantigens from the autograft. In certain embodiments, the previous graft is an autologous cell, tissue, and/or organ. In some embodiments, the susceptible patient has previously received allogeneic CAR-T cell-based therapy or autologous CAR-T cell-based therapy. Non-limiting examples of autologous CAR-T cell-based therapies include bucoba Ji Aolun race Sicospiro->Ai Kaba Ji Weisai->Li Jimai Racing malasseziaSha Jinlu->Descartes-08 and Descartes-11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, and AUTO4 from Autolus Limited. Non-limiting examples of allogeneic CAR-T cell-based therapies include UCARTCS from Cellectis, PBCAR19B and PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics, and CYAD-211 from Clyad oncogy. In some embodiments, the susceptible patient is administered a second therapy comprising cells of the present technology after the patient has previously received a first therapy comprising allogeneic CAR-T cell-based therapy or autologous CAR-T cell-based therapy (excluding cells of the present technology). In some embodiments, after the patient has previously received the first and/or second therapies comprising allogeneic CAR-T cell-based therapies or autologous CAR-T cell-based therapies (excluding cells of the present technology), the susceptible patient is then administered a third therapy comprising cells of the present technology. In some embodiments, after the patient has previously received a series of therapies comprising allogeneic CAR-T cell-based therapies or autologous CAR-T cell-based therapies (excluding cells of the present technology), the sensitive patient is then administered a subsequent therapy comprising cells of the present technology. In some embodiments, (i) after a failed treatment such as, but not limited to, an allogeneic or autologous CAR-T cell-based therapy that does not include the cells provided herein, (ii) after a therapeutically ineffective treatment such as, but not limited to, an allogeneic or autologous CAR-T cell-based therapy that does not include the cells provided herein, or (iii) after a therapeutically effective treatment such as, but not limited to, an allogeneic or autologous CAR-T cell-based therapy that does not include the cells provided herein (in some examples, in one In some embodiments, including subsequent treatments of the first line, the second line, the third line, and the additional line), the methods provided herein are useful as an on-line treatment for a particular disorder or disease.

In certain embodiments, a susceptible patient suffers from an allergy and is sensitive to one or more allergens. In the illustrated embodiment, the patient suffers from hay fever, food allergy, insect allergy, drug allergy, and/or atopic dermatitis.

Any suitable method known in the art may be used in determining whether a patient is a sensitive patient in view of the present disclosure. Examples of methods for determining whether a patient is a sensitive patient include, but are not limited to, cell-based assays, including complement dependent cell killing (CDC) and flow cytometry assays, and solid phase assays, including ELISA and polystyrene bead-based array assays. Other examples of methods for determining whether a patient is a sensitive patient include, but are not limited to, antibody screening methods, percent disc-reactive antibody (PRA) assays, luminex-based assays, e.g., using Single Antigen Beads (SAB) and Luminex IgG assays, assessing the Mean Fluorescence Intensity (MFI) value of HLA antibodies, computing disc-reactive antibody (cPRA) assays, igG titration tests, complement fixation assays, igG subtype assays, and/or in Colvin et al, circulation.2019mar 19;139 (12): e553-e 578.

In some embodiments, the patient undergoing treatment with the subject low immunity cells receives prior treatment. In some embodiments, the same disorder as the previous treatment is treated using low immunity cells. In some embodiments, the low immunity cell therapy is used for a condition that is different from the previous therapy. In some embodiments, the low-immune cells administered to the patient exhibit enhanced therapeutic effects to treat the same condition or disease that was previously treated. In some embodiments, the administered low-immunity cells exhibit a longer therapeutic effect on the treatment of a disorder or disease in a patient than prior treatments. In exemplary embodiments, the administered cells exhibit enhanced potential, efficacy, and/or specificity for cancer cells as compared to prior treatments. In certain embodiments, the low immunity cell is a CAR-T-cell to treat cancer.

In some embodiments, the methods provided herein can be used as a next on-line therapy for a particular disorder or disease after failed therapy, after therapy-ineffective therapy, or after effective therapy, including in each case after first line, second line, third line, and additional line therapy. In some embodiments, the prior treatment (e.g., first line treatment) is a treatment that is not therapeutically effective. As used herein, a "treatment ineffective" treatment refers to a treatment that produces less than desirable clinical results in a patient. For example, for the treatment of a cellular defect, a therapeutically ineffective treatment may refer to the following treatments: functional cells and/or cellular activities that do not reach the desired level replace defective cells in the patient, and/or lack therapeutic persistence. For cancer treatment, treatment that is not therapeutically effective refers to treatment that does not reach the desired level of potential, efficacy, and/or specificity. The therapeutic effect may be measured using any suitable technique known in the art. In some embodiments, the patient responds to prior therapy. In some embodiments, the prior treatment is a cell, tissue, and/or organ transplant that is rejected by the patient. In some embodiments, the prior treatment comprises a mechanically assisted treatment. In some embodiments, the mechanically assisted treatment comprises hemodialysis or ventricular assist devices. In some embodiments, the patient responds to the mechanical assist therapy. In some embodiments, the prior treatment includes a population of therapeutic cells that includes a safety switch that results in death of the therapeutic cells in the event that they grow and divide in an undesired manner when the safety switch is activated. In some embodiments, the patient develops an immune response because of the safety switch-induced therapeutic cell death. In some embodiments, the patient is sensitive to previous treatments. In the illustrated embodiment, the patient is not sensitive due to the low immunity cells administered.

In some embodiments, the subject low immunity cells are administered prior to, concurrently with, and/or after providing the tissue, organ, and/or partial organ transplant to a patient in need thereof. In some embodiments, the patient does not exhibit an immune response to the low immunity cells. In some embodiments, the low immunity cells are administered to a patient for treating a cell defect in a particular tissue and/or organ and the patient subsequently receives a tissue or organ transplant for the same particular tissue or organ. In some embodiments, the low immunity cells are administered to the patient in situ in the tissue or organ for transplantation. In some embodiments, the low immunity cells are administered to the patient in situ in the tissue or organ, either before or after the tissue or organ transplant. In this embodiment, the hypoimmunity cell therapy acts as a bridging therapy to the final tissue or organ replacement. For example, in some embodiments, the patient has a liver disorder and receives the low immunity hepatocyte therapy provided herein prior to receiving the liver transplant. In some embodiments, the patient has a liver disorder and receives a low immunity hepatocyte treatment as provided herein after receiving the liver transplant. In some embodiments, the low immunity cells are administered to a patient to treat a cellular defect in a particular tissue and/or organ and the patient subsequently receives a tissue and/or organ transplant for a different tissue or organ. For example, in some embodiments, the patient is a diabetic patient treated with low-immunity pancreatic beta cells prior to receiving the kidney transplant. In some embodiments, the patient is a diabetic patient treated with low-immunity pancreatic beta cells after receiving the kidney transplant. In some embodiments, the low immunity cell therapy is administered to the donor tissue and/or organ before and/or after the patient receives the tissue or organ transplant. In some embodiments, the method is for treating a cellular defect. In exemplary embodiments, the tissue or organ graft is a heart graft, a lung graft, a kidney graft, a liver graft, a pancreas graft, an intestinal graft, a stomach graft, a cornea graft, a bone marrow graft, a vascular graft, a heart valve graft, and/or a bone graft.

Methods of treating patients generally are by administering cells, particularly the low immunity cells provided herein. It will be appreciated that for all of the various embodiments described herein that relate to cells and/or timing of treatment, administration of cells is accomplished by a method or pathway that results in at least partial localization of the introduced cells at the desired site. The cells may be implanted directly into the desired site or administered by any suitable route that results in delivery to the desired location in the subject, wherein at least a portion of the implanted cells or components of the cells remain viable. In some embodiments, the cells are implanted in situ at a desired organ or desired location of the organ. In some embodiments, cells may be implanted into donor tissue and/or organ before and/or after the patient receives the tissue or organ transplant. In some embodiments, the cells are administered to treat a disease or disorder, such as any disease, disorder, condition, and/or symptom thereof that can be alleviated by cell therapy.

In some embodiments, the population of cells is administered for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, or at least 1 month or more after the patient is sensitive. In some embodiments, the population of cells is administered for at least 1 week (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or more) or longer after the patient is sensitive or exhibits sensitive characteristics or features. In some embodiments, the population of cells is administered for at least 1 month (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months or more) or more after the patient has received the graft (e.g., allograft), has been pregnant (e.g., had or had alloimmunity at pregnancy), and/or is sensitive and/or exhibits sensitive characteristics and/or features.

In some embodiments, a patient who has received a graft, who has been pregnant (e.g., has or has had alloimmunity at pregnancy), and/or who is susceptible to an antigen (e.g., an alloantigen) is administered a dosing regimen comprising a first dose of a population of cells described herein, a recovery period after the first dose, and a second dose of a population of cells described. In some embodiments, the complex of cell types present in the first population of cells is different from the second population of cells. In certain embodiments, the complexes of cell types present in the first population of cells and the second population of cells are the same or substantially equal. In many embodiments, the first population of cells and the second population of cells comprise the same cell type. In some embodiments, the first population of cells and the second population of cells comprise different cell types. In some embodiments, the first population of cells comprises the same percentage of cell types as the second population of cells. In other embodiments, the first population of cells and the second population of cells comprise different percentages of cell types.

In some embodiments, the population of cells is administered for the treatment of a cell defect and/or as a cell therapy for the treatment of a disorder or disease in a tissue and/or organ selected from the group consisting of: heart, lung, kidney, liver, pancreas, intestine, stomach, cornea, bone marrow, blood vessels, heart valves, brain, spinal cord and/or bone.

In some embodiments, the cell defect is associated with a neurodegenerative disease and the cell therapy is used to treat the neurodegenerative disease. In some embodiments, the neurodegenerative disease is selected from the group consisting of: leukodystrophy, huntington's disease, parkinson's disease, multiple sclerosis, transverse myelitis, and/or pemphigus disease (PMD). In some embodiments, the cell is selected from the group consisting of: glial progenitor cells, oligodendrocytes, astrocytes and dopamine neurons, optionally, wherein the dopamine neurons are selected from the group consisting of: neural stem cells, neural progenitor cells, immature dopamine neurons, and mature dopamine neurons. In some embodiments, the cellular defect is associated with a liver disease and the cell therapy is used to treat the liver disease. In some embodiments, the liver disease comprises cirrhosis of the liver. In some embodiments, the cell is a hepatocyte or hepatic progenitor cell. In some embodiments, the cellular defect is associated with a corneal disease and the cell therapy is used to treat the corneal disease. In some embodiments, the corneal disease is Fuchs dystrophy or congenital genetic endothelial dystrophy. In some embodiments, the cell is a corneal endothelial progenitor cell or a corneal endothelial cell. In some embodiments, the cell defect is associated with a cardiovascular disorder or disease and the cell therapy is used to treat the cardiovascular disorder or disease. In some embodiments, the cardiovascular disease is myocardial infarction and/or congestive heart failure. In some embodiments, the cell is a cardiomyocyte or cardiac progenitor. In some embodiments, the cellular defect is associated with diabetes and the cell therapy is used to treat diabetes. In some embodiments, the cell is a pancreatic islet cell, including a pancreatic beta islet cell, optionally wherein the pancreatic islet cell is selected from the group consisting of: pancreatic islet progenitor cells, immature pancreatic islet cells, and mature pancreatic islet cells. In some embodiments, the cell defect is associated with a vascular disorder or disease and the cell therapy is used to treat the vascular disorder or disease. In some embodiments, the cell is an endothelial cell. In some embodiments, the cell deficiency is associated with autoimmune thyroiditis and the cell therapy is used to treat autoimmune thyroiditis. In some embodiments, the cell is a thyroid progenitor cell. In some embodiments, the cell deficiency is associated with kidney disease and the cell therapy is used to treat kidney disease. In some embodiments, the cell is a kidney precursor cell or a kidney cell.

In some embodiments, the population of cells is administered to treat cancer. In some embodiments, the population of cells is administered to treat cancer and the population of cells is a population of CAR-T cells. In some embodiments, the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

In some embodiments, the patient is receiving a tissue or organ transplant, optionally, wherein the tissue or organ transplant or partial organ transplant is selected from the group consisting of: heart grafts, lung grafts, kidney grafts, liver grafts, pancreas grafts, intestinal grafts, stomach grafts, cornea grafts, bone marrow grafts, vascular grafts, heart valve grafts, bone grafts, partial lung grafts, partial kidney grafts, partial liver grafts, partial pancreas grafts, partial intestinal grafts and/or partial cornea grafts.

In some embodiments, the tissue or organ graft is an allograft graft. In some embodiments, the tissue or organ graft is an autograft graft. In some embodiments, the population of cells is administered to treat a cell defect in a tissue or organ and the tissue or organ transplant is a replacement for the same tissue or organ. In some embodiments, the population of cells is administered to treat a cellular defect in a tissue and/or organ and the tissue and/or organ graft is a replacement for a different tissue or organ. In some embodiments, the organ transplant is a kidney transplant and the population of cells is a population of kidney precursor cells or kidney cells. In some embodiments, the patient has diabetes and the population of cells is a population of beta islet cells. In some embodiments, the organ transplant is a heart transplant and the population of cells is a population of heart progenitor cells or paced cells. In some embodiments, the organ transplant is a pancreatic transplant and the population of cells is a population of pancreatic beta islet cells. In some embodiments, the organ transplant is a partial liver transplant and the population of cells is a population of hepatocytes or hepatic progenitors.

In some embodiments, the first administration of a population of low immunity cells during recovery begins after and ends when such cells are no longer present or undetectable in the patient. In some embodiments, the duration of the recovery period is at least 1 week (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or more) or more after the initial administration of the cells. In some embodiments, the duration of the recovery period is at least 1 month (e.g., 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, or more) or longer after the initial administration of the cells.

In some embodiments, the administered population of low immune cells causes a reduced or reduced level of systemic TH1 activation in the patient. In some examples, the level of systemic TH1 activation by the cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% less than the level of systemic TH1 activation produced by administration of the immune cell. In some embodiments, the administered population of low immune cells does not cause systemic TH1 activation in the patient.

In some embodiments, the administered population of low immunity cells causes a reduced or reduced level of immune activation of Peripheral Blood Mononuclear Cells (PBMCs) in the patient. In some examples, the level of immune activation of PBMCs by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% less than the level of immune activation of PBMCs produced by administration of the immune cells. In some embodiments, the population of low immunity cells administered is not in the patient that causes immune activation of PBMCs.

In some embodiments, the administered population of low immunity cells causes reduced or decreased levels of donor-specific IgG antibodies in the patient. In some examples, the level of donor-specific IgG antibodies elicited by the cells is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% lower than the level of donor-specific IgG antibodies produced by administration of the immune cells. In some embodiments, the administered population of low immunity cells does not elicit donor-specific IgG antibodies in the patient.

In some embodiments, the administered population of low immunity cells causes reduced or reduced levels of IgM and IgG antibody production in the patient. In some examples, the level of IgM and IgG antibody production by the cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% less than the level of IgM and IgG antibody production produced by administration of the immune cell. In some embodiments, the administered population of low immunity cells does not elicit IgM and IgG antibody production in the patient.

In some embodiments, the administered population of low immunity cells causes a reduced or decreased level of cytotoxic T cell killing in the patient. In some examples, the level of cytotoxic T cell killing by the cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% less than the level of cytotoxic T cell killing produced by administration of the immune cell. In some embodiments, the administered population of low immunity cells does not cause cytotoxic T cell killing in the patient.

As discussed above, provided herein are, in certain embodiments, cells that can be administered to a patient susceptible to an alloantigen, such as a human leukocyte antigen. In some embodiments, the patient is or has become pregnant, e.g., has an alloimmune effect (e.g., hemolytic disease of the fetus and neonate (HDFN), neonatal Alloimmune Neutropenia (NAN), or fetal and neonatal alloimmune thrombocytopenia (FNAIT)) at the time of pregnancy. In other words, the patient suffers from or has suffered from disorders or conditions associated with alloimmunity during pregnancy, such as, but not limited to, hemolytic Disease (HDFN) of the fetus and neonate, neonatal Alloimmune Neutropenia (NAN), and fetal and neonatal alloimmune thrombocytopenia (FNAIT). In some embodiments, the patient has received an allograft, such as, but not limited to, an allograft, an allotransfusion, an allograft, or an allograft. In some embodiments, the patient presents memory B cells to the alloantigen. In some embodiments, the patient presents memory T cells to the alloantigen. This patient may present both memory B and memory T cells to alloantigens.

Upon administration of the cells, the patient exhibits no systemic immune response or a reduced level of systemic immune response compared to a response to cells without low immunity. In some embodiments, the patient exhibits an adaptive immune response or a reduced level of adaptive immune response compared to a response to cells without low immunity. In some embodiments, the patient exhibits no or reduced levels of innate immune response as compared to response to cells without low immunity. In some embodiments, the patient exhibits no T cell response or a reduced level of T cell response compared to a response to cells without low immunity. In some embodiments, the patient exhibits a B-cell-free response or a reduced level of B-cell response compared to a response to cells without low immunity.

As described in further detail herein, provided herein are a population of low immunity cells comprising an exogenous CD47 polypeptide and a reduced expression of MHC class I human leukocyte antigen, a population of low immunity cells comprising an exogenous CD47 polypeptide and a reduced expression of MHC class II human leukocyte antigen, and a population of low immunity cells comprising an exogenous CD47 polypeptide and a reduced expression of MHC class I and II human leukocyte antigen.

B. Low immunity cells

Provided herein are methods comprising modulating expression of MHC I molecules, MHC II molecules, or MHC I and MHC II moleculesA modified cell of the target polynucleotide sequence. In certain aspects, the modification comprises increased expression of CD 47. In some embodiments, the cell includes one or more transient or genomic modifications that reduce expression of MHC class I molecules and modifications that increase expression of CD 47. In other words, the engineered cells comprise exogenous polynucleotides encoding CD47 protein and exhibit reduced or silenced surface expression of one or more MHC class I molecules. In some embodiments, the cell includes one or more genomic modifications that reduce expression of MHC class II molecules and modifications that increase expression of CD 47. In some examples, the engineered cells comprise exogenous CD47 nucleic acids and proteins and surface expression of one or more MHC class I molecules that exhibit reduced or silencing. In some embodiments, the cell comprises one or more genomic modifications that reduce or eliminate expression of MHC class II molecules, and a modification that increases expression of CD 47. In some embodiments, the engineered cells comprise exogenous CD47 protein, exhibit reduced or silenced surface expression of one or more MHC class I molecules, and exhibit reduced or absent surface expression of one or more MHC class II molecules. In many embodiments, the cell is B2M ^indel/indel 、CIITA ^indel/indel CD47tg cells.

The reduction of MHC I and/or MHC II expression may be accomplished, for example, by one or more of the following: (1) Direct targeting polymorphic HLA-pair genes (HLA-A, HLA-B, HLA-C) and MHC-II genes; (2) Removal of B2M, which would reduce surface transport of all MHC-I molecules; and/or (3) deleting one or more components of MHC enhancers, such as LRC5, RFX-5, RFXANK, RFXAP, IRFl, NF-Y (including NFY-A, NFY-B, NFY-C) and CIITA, which are important for HLA expression.

In certain embodiments, HLA expression is disrupted. In some embodiments, HLA expression is interfered with by targeting individual HLA (e.g., knockout expression of HLA-a, HLA-B, and/or HLA-C), targeting transcriptional modulators of HLA expression (e.g., knockout expression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, and/or IRF-1), blocking surface transport of MHC class I molecules (e.g., knockout expression of B2M and/or TAP 1), and/or targeting HLA-razors (see, e.g., WO 2016183041).

In certain aspects, cells disclosed herein, including stem cells or differentiated stem cells, do not express one or more human leukocyte antigens (e.g., HLA-A, HLa-B, and/or HLa-C) corresponding to MHC-I and/or MHC-II and are therefore characterized by low immunity. For example, in certain aspects, cells disclosed herein comprising stem cells or differentiated stem cells have been modified such that the stem cells or differentiated stem cells thus prepared do not express or exhibit reduced expression of one or more of the following MHC-I molecules: HLA-A, HLA-B and HLA-C. In some embodiments, one or more of HLA-A, HLA-B, and HLA-C may be "knocked out" by cells. Cells with knockdown HLA-A genes, HLA-B genes, and/or HLA-C genes may exhibit reduced or eliminated expression of each knockdown gene.

In certain embodiments, guide RNAs that allow for simultaneous deletion of all MHC class I dual genes by targeting a reserved region in an HLA gene are identified as HLA razors. In some embodiments, the guide RNA is part of a CRISPR system, e.g., a CRISPR-Cas9 system. In an alternative aspect, the gRNA is part of a TALEN system. In one aspect, WO2016183041 describes an HLA Razor targeting an identified retention region in an HLA. In other aspects, multiple HLA razors are used that target the identified retention regions. In general it is understood that any guidance targeting a retention region in an HLA can be used as an HLA Razor.

In some embodiments, the cell comprises a modification to increase expression of CD47 and one or more factors selected from the group consisting of: DUX4, CD24, CD27, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8, CD16, CD52, H2-M3 and Serphinb 9.

In some embodiments, the cell comprises genomic modifications of one or more polynucleotide sequences of interest that regulate expression of MHC class I molecules, MHC class II molecules, or MHC class I and MHC class II molecules. In some embodiments, a gene editing system is used to modify one or more polynucleotide sequences of interest. In some embodiments, the targeted polynucleotide sequence is one or more selected from the group consisting of: including B2M, CIITA and NLRC5. In some embodiments, the cell comprises a gene editing modification to the B2M gene. In some embodiments, the cell comprises a gene editing modification to the CIITA gene. In some embodiments, the cell comprises a gene editing modification to the NLRC5 gene. In some embodiments, the cells comprise gene editing modifications to B2M and CIITA genes. In some embodiments, the cell comprises a gene editing modification to the B2M and NLRC5 genes. In some embodiments, the cells comprise gene editing modifications to the CIITA and NLRC5 genes. In a particular embodiment, the cell comprises a gene editing modification to the B2M, CIITA and NLRC5 genes. In some embodiments, the genome of the cell has been altered to reduce or delete an important component of HLA expression.

In some embodiments, the disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary cell, or a CAR-T cell) or population thereof comprising a contiguously stretched genome in which a gene has been edited to delete genomic DNA, thereby reducing or eliminating expression of an MHC class I molecule in the cell or population thereof, e.g., surface expression of an MHC class I molecule in the cell or population thereof. In certain aspects, the disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary cell, or a CAR-T cell), or a population thereof, comprising a contiguously stretched genome in which a gene has been edited to delete genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof. In a particular aspect, the disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary cell, or a CAR-T cell), or a population thereof, comprising a contiguously stretched genome in which one or more genes have been edited to delete genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules in the cell or population thereof.

In certain embodiments, expression of MHC I molecules and/or MHC II molecules is modulated by targeting and deleting contiguous stretches of genomic DNA, thereby reducing or eliminating expression of a gene of interest selected from the group consisting of: B2M, CIITA and NLRC5. In some embodiments, described herein are genetically edited cells (e.g., modified human cells) that contain exogenous CD47 protein and an unactivated or modified CIITA gene sequence and, in some examples, additional genetic modifications that do not activate or modify a B2M gene sequence. In some embodiments, described herein are genetically edited cells comprising an exogenous CD47 protein and an unactivated or modified CIITA gene sequence and, in some examples, additional genetic modifications that do not activate or modify the NLRC5 gene sequence. In some embodiments, described herein are genetically edited cells comprising exogenous CD47 protein and an unactivated or modified B2M gene sequence and, in some examples, additional genetic modifications that do not activate or modify the NLRC5 gene sequence. In some embodiments, described herein are genetically edited cells comprising exogenous CD47 protein and an unactivated or modified B2M gene sequence and, in some examples, additional genetic modifications of the unactivated or modified CIITA gene sequence and the NLRC5 gene sequence.

In some embodiments, the cell is B2M ^-/- 、CIITA ^-/- 、TRAC ^-/- 、TRB ^-/- CD47tg cells. In some embodiments, B2M ^-/- 、CIITA ^-/- 、TRAC ^-/- 、TRB ^-/- CD47tg cells are primary T cells or T cells derived from low immunity pluripotent cells (e.g., low immunity ipscs).

In some embodiments, the cell is B2M ^-/- 、CIITA ^-/- 、TRAC ^-/- And CD47tg cells. In some embodiments, B2M ^-/- 、CIITA ^-/- 、TRAC ^-/- And CD47tg cells are primary T cells or T cells derived from low immunity pluripotent cells (e.g., low immunity ipscs).

In some embodiments, the cells described herein include, but are not limited to, pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from or produced from such stem cells, hematopoietic stem cells, primary T cells, chimeric Antigen Receptor (CAR) T cells, and any progeny thereof.

In some embodiments, the primary T cell is selected from the group consisting of: cytotoxic T-cells, helper T-cells, memory T-cells, regulatory T-cells, tumor infiltrating lymphocytes, and combinations thereof.

In some embodiments, the low immune T cells and primary T cells overexpress CD47 and Chimeric Antigen Receptor (CAR), and include genomic modifications of the B2M gene. In some embodiments, the low immune T cells and the primary T cells overexpress CD47 and include genomic modifications of the CIITA gene. In some embodiments, the low immune T cells and primary T cells overexpress CD47 and CAR, and include genomic modifications of the TRAC gene. In some embodiments, the low immune T cells and primary T cells overexpress CD47 and CAR, and include genomic modifications of the TRB gene. In some embodiments, the low immune T cells and primary T cells overexpress CD47 and CAR, and comprise one or more genomic modifications selected from the group consisting of: B2M, CIITA, TRAC and TRB genes. In some embodiments, the low immune T cells and primary T cells overexpress CD47 and CAR, and include genomic modifications of B2M, CIITA, TRAC and TRB genes. In some embodiments, the cell is a B2M that also expresses a CAR ^-/- 、CIITA ^-/- 、TRAC ^-/- And CD47tg cells.

In some embodiments, the cell is a B2M that also expresses a CAR ^-/- 、CIITA ^-/- 、TRB ^-/- And CD47tg cells. In some embodiments, the cell is a B2M that also expresses a CAR ^-/- 、CIITA ^-/- 、TRAC ^-/- 、TRB ^-/- And CD47tg cells. In many embodiments, the cell is a B2M that also expresses a CAR ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel And CD47tg cells. In many embodiments, the cell is a B2M that also expresses a CAR ^indel/indel 、CIITA ^indel/indel 、TRB ^indel/indel And CD47tg cells. In many embodiments, the cell is a B2M that also expresses a CAR ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel 、TRB ^indel/indel And CD47tg cells. In some embodiments, the modified cell is a pluripotent stem cell, an induced pluripotent stem cell, a cell differentiated from such pluripotent stem cell and induced pluripotent stem cell, or a primary T cell. Non-limiting examples of primary T cells include cd3+ T cells, cd4+ T cells, cd8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, th1 cells, th2 cells, th9 cells, th17 cells, T-follicular helper (Tfh) cells, cytotoxic T Lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA) cells, tissue resident memory (Trm) cells, virtual memory T cells, congenital memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cells. In some embodiments, the primary T cell is selected from the group consisting of: cytotoxic T-cells, helper T-cells, memory T-cells, regulatory T-cells, tumor infiltrating lymphocytes, and/or combinations thereof.

In some embodiments, the primary T cell is a primary T cell pool from one or more donor subjects other than the recipient subject (e.g., the patient to whom the cell is administered). Primary T cells can be obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100 or more donor subjects and pooled together. Primary T cells may be obtained from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more 20 or more, 50 or more, or 100 or more donor subjects and pooled together. In some embodiments, the primary T cells are obtained from one or more subjects, and in some examples, the primary T cells or primary T cell pools are in vitro cultured. In some embodiments, the primary T cells or primary T cell pools are engineered to exogenously express CD47 and cultured in vitro.

In some embodiments, the primary T cell or primary T cell pool is engineered to express a Chimeric Antigen Receptor (CAR). The CAR may be any known to those of ordinary skill in the art to which the invention pertains. Useful CARs include those that bind an antigen selected from the group consisting of: CD19, CD20, CD22, CD38, CD123, CD138 and BCMA. In some examples, the CAR is the same or equivalent to that used in FDA-approved CAR-T cell therapies, such as, but not limited to, bucoba Ji Aolun, cicasirox, ai Kaba Ji Weisai, li Jimai rassmaracer, ti Sha Jinlu, or other that used in clinical trials in the recipient study.

In some embodiments, the primary T cell or primary T cell pool is engineered to exhibit reduced expression of an endogenous T cell receptor as compared to an unmodified primary T cell. In some embodiments, the primary T cell or primary T cell pool is engineered to exhibit reduced expression of CTLA4, PD1, or both CTLA4 and PD1 as compared to an unmodified primary T cell. Methods for genetically modifying cells including T cells are described in detail in, for example, WO2020018620 and WO2016183041, the disclosures of which are incorporated herein by reference in their entirety, including tables, appendages, sequence listings and figures.

In some embodiments, the CAR-T cell comprises a CAR selected from the group consisting of: (a) A first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) A second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) A third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene following successful signaling of the CAR.

In some embodiments, the CAR-T cell comprises a CAR comprising an antigen binding domain, a transmembrane, and one or more signaling domains. In some embodiments, the CAR also comprises a linker. In some embodiments, the CAR comprises a CD19 antigen binding domain. In some embodiments, the CAR comprises a CD28 or CD8 a transmembrane domain. In some embodiments, the CAR comprises a CD8 a signal peptide. In some embodiments, the CAR comprises a Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 14). In some embodiments, the antigen binding domain of the CAR is selected from the group including, but not limited to, (a) the antigen binding domain targets an antigenic property of a tumor cell; (b) An antigen binding domain that targets an antigenic feature of a T cell; (c) The antigen binding domain targets an antigenic feature of an autoimmune or inflammatory disorder; (d) An antigen binding domain that targets an antigenic feature of senescent cells; (e) An antigen binding domain that targets an antigenic feature of an infectious disease; and (f) an antigen binding domain that binds to a cell surface antigen of a cell.

In some embodiments, the antigen binding domain is selected from the group consisting of: antibodies, antigen binding portions or fragments thereof, scFv and Fab. In some embodiments, the antigen binding domain binds to CD19, CD20, CD22, CD38, CD123, CD138, or BCMA. In some embodiments, the antigen binding domain is an anti-CD 19 scFv, such as, but not limited to FMC63.

In some embodiments, the transmembrane domain comprises one selected from the group consisting of: the transmembrane region of tcra, tcrβ, tcrζ, cd3ζ, cd3γ, cd3δ, cd3ζ, CD4, CD5, cd8α, cd8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40L/CD154, CD45, CD64, CD80, CD86, OX40/CD134, 4-1BB/CD137, CD154, fcsry, VEGFR2, FAS, FGFR2B and functional variants thereof.

In some embodiments, the signaling domain of the CAR comprises a co-stimulatory domain. For example, the signaling domain may comprise a co-stimulatory domain. Alternatively, the signaling domain may comprise one or more co-stimulatory domains. In certain embodiments, the signaling domain comprises one co-stimulatory domain. In other embodiments, the signaling domain comprises a plurality of co-stimulatory domains. In some examples, when the CAR comprises two or more co-stimulatory domains, the two co-stimulatory domains are not identical. In some embodiments, the co-stimulatory domain comprises two co-stimulatory domains that are not identical. In some embodiments, the one co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation. In some embodiments, the plurality of co-stimulatory domains enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation.

As described herein, the fourth generation CAR can comprise an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of cytokine genes following successful signaling of the CAR. In some examples, the cytokine gene is an endogenous or exogenous cytokine gene of the low immunity cell. In some examples, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma and functional fragments thereof. In some embodiments, the domain that induces expression of the cytokine gene upon successful signaling of the CAR comprises a transcription factor or functional domain or fragment thereof.

In some embodiments, the CAR comprises a CD3 zeta (cd3ζ) domain or an immune receptor (immune receptor) tyrosine-based activation motif (ITAM) or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immune receptor tyrosine based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In other embodiments, the CAR comprises (i) a CD3 zeta domain or an immune receptor tyrosine based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zet a domain or an immune receptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes. In some embodiments, the CAR comprises (i) an anti-CD 19 scFv; (ii) A CD8 a hinge and a transmembrane domain or functional variant thereof; (iii) a 4-1BB co-stimulatory domain or a functional variant thereof; and (iv) a CD3 zeta signaling domain or a functional variant thereof.

Methods for introducing CAR constructs or producing CAR-T cells are known to those of ordinary skill in the art of the invention. The detailed description can be found, for example, in Vormittag et al, curr Opin Biotechnol.,2018,53,162-181; and Eyquem et al Nature,2017,543,113-117.

In some embodiments, cells derived from primary T cells comprise an endogenous T cell receptor that reduces expression, e.g., by disrupting an endogenous T cell receptor gene (e.g., T cell receptor alpha constant region (referred to as "TRAC") and/or T cell receptor beta constant region (referred to as "TRBC" or "TRB"). In some embodiments, an exogenous nucleic acid encoding a polypeptide disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted into the disrupted T cell receptor gene.

In some embodiments, the cells derived from primary T cells comprise reduced expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA 4) and/or programmed cell death (PD 1). Methods of reducing or eliminating CTLA4, PD1, and both CTLA4 and PD1 expression can include any method recognized by those of ordinary skill in the art of the invention, such as, but not limited to, genetic modification techniques utilizing rare-cutting endonucleases with RNA silencing or RNA interference techniques. Non-limiting examples of rare-cutting endonucleases include any Cas protein, TALEN, zinc finger nuclease, meganuclease, and/or homing endonuclease. In some embodiments, an exogenous nucleic acid encoding a polypeptide disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic agent disclosed herein) is inserted into the CTLA4 and/or PD1 gene locus.

In some embodiments, the CD47 transgene is inserted into a preselected locus of the cell. In some embodiments, the transgene encodes insertion of the CAR into a preselected locus of the cell. In many embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into preselected loci of the cell. The preselected locus may be a safe harbor locus or a target locus. Non-limiting examples of safe harbor loci include, but are not limited to, the CCR5 locus, PPP1R12C (also known as AAVS 1) locus, CLYBL locus and/or Rosa locus (e.g., rosa26 locus). Non-limiting examples of loci of interest include, but are not limited to, CXCR4 genes, albumin genes, SHS231 loci, F3 genes (also known as CD 142), MICA genes, MICB genes, LRP1 genes (also known as CD 91), HMGB1 genes, ABO genes, RHD genes, FUT1 genes, KDM5D genes (also known as HY), B2M genes, CIITA genes, TRAC genes, TRBC genes, CCR5 genes, F3 (i.e., CD 142) genes, MICA genes, MICB genes, LRP1 genes, HMGB1 genes, ABO genes, RHD genes, FUT1 genes, KDM5D (i.e., HY) genes, PDGFRa genes, OLIG2 genes, and/or GFAP genes. In some embodiments, the preselected locus is selected from the group consisting of: B2M locus, CIITA locus, TRAC locus and TRB locus. In some embodiments, the preselected locus is a B2M locus. In some embodiments, the preselected locus is a CIITA locus. In some embodiments, the preselected locus is the TRAC locus. In some embodiments, the preselected locus is a TRB locus.

In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the same locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into different loci. In many instances, the CD47 transgene is inserted into a safe harbor or target locus. In many instances, the transgenic encoded CAR is inserted into a safe harbor or target locus. In some examples, the CD47 transgene is inserted into the B2M locus. In some examples, the transgene encodes insertion of the CAR into the B2M locus. In some embodiments, the CD47 transgene is inserted into the CIITA locus. In some embodiments, the transgene encodes insertion of the CAR into the CIITA locus. In some embodiments, the CD47 transgene is inserted into the TRAC locus. In some embodiments, the transgene encodes insertion of the CAR into the TRAC locus. In other embodiments, the CD47 transgene is inserted into the TRB locus. In other embodiments, the transgene encodes insertion of the CAR into the TRB locus. In some embodiments, the CD47 transgene and the CAR-encoding transgene are inserted into a safe harbor locus (e.g., CCR5 locus, PPP1R12C locus, CLYBL locus, and/or Rosa locus).

In many embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into a safe harbor or locus of interest. In many embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into a safe harbor or locus of interest. In many embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into a safe harbor or target locus. In many embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the TRAC locus. In many embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the TRAC locus. In many embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the TRAC locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the TRB locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the B2M locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the B2M locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the B2M locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the CIITA locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the CIITA locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the CIITA locus. In some examples, the promoter that controls expression of any of the transgenes is a constitutive promoter. In other examples, the promoter for any of the transgenes is an inducible promoter. In some embodiments, the promoter is an EF1 alpha (EF 1 alpha) promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, both the CD47 transgene and the transgene encoding the CAR are under the control of a constitutive promoter. In some embodiments, both the CD47 transgene and the transgene encoding the CAR are under the control of an inducible promoter. In some embodiments, the CD47 transgene is under the control of a constitutive promoter and the transgene encoding the CAR is under the control of an inducible promoter. In some embodiments, the CD47 transgene is under the control of an inducible promoter and the transgene encoding the CAR is under the control of a constitutive promoter. In various embodiments, the CD47 transgene is under the control of the EF1 alpha promoter and the transgene encoding the CAR is under the control of the EF1 alpha promoter. In other embodiments, expression of both the CD47 transgene and the transgene encoding the CAR is under the control of a single EF1 alpha promoter. In various embodiments, the CD47 transgene is under the control of a CAG promoter to encode a transgene of the CAR. In other embodiments, the expression of both the CD47 transgene and the transgene encoding the CAR is controlled by a single CAG promoter. In some embodiments, the CD47 transgene is under the control of the CAG promoter and the transgene encoding the CAR is under the control of the EF1 alpha promoter. In some embodiments, the CD47 transgene is under the control of the EF1 alpha promoter and the transgene encoding the CAR is under the control of the CAG promoter.

In some embodiments, the cells described herein comprise a safety switch. The term "safety switch" as used herein refers to a system that controls the expression of a gene or protein of interest, which when down-regulated or up-regulated, results in cell clearance or death, e.g., by recognition of the host immune system. The safety switch may be designed to be triggered by an exogenous molecule upon occurrence of an adverse clinical event. Safety switches can be engineered by modulating expression at the DNA, RNA and protein levels. The safety switch includes a protein or molecule that allows control of cellular activity in response to an adverse event. In one embodiment, the safety switch is a "kill switch" which is expressed in an inactive state and is fatal to the cell expressing the safety switch when the switch is activated by a selective, externally provided agent. In one embodiment, the safety switch gene is cis-acting in relation to the gene of interest in the construct. Activation of the safety switch causes the cell to kill itself only or to kill itself and neighboring cells by apoptosis or necrosis. In some embodiments, a cell described herein, e.g., a stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a primary cell, or a differentiated cell, including, but not limited to, a cardiac cell, a cardiac progenitor, a neural cell, a glial progenitor, an endothelial cell, a T cell, a B cell, a pancreatic islet cell, a retinal pigment epithelial cell, a hepatic cell, a thyroid cell, a skin cell, a blood cell, a plasma cell, a platelet, a kidney cell, an epithelial cell, a CART cell, an NK cell, and/or a CAR-NK cell, comprises a safety switch.

In some embodiments, the cells described herein comprise a "suicide gene" (or "suicide switch"). Suicide genes can lead to low immune cell death if the low immune cells grow and divide in an undesirable manner. Suicide gene ablation methods include suicide genes in gene transfer vectors encoding proteins that cause cell killing only when specific compounds are activated. Suicide genes may encode enzymes that selectively convert non-toxic compounds to highly toxic metabolites. In some embodiments, a cell described herein, e.g., a stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a primary cell or a differentiated cell, including, but not limited to, a cardiac cell, a cardiac progenitor, a neural cell, a glial progenitor, an endothelial cell, a T cell, a B cell, a pancreatic islet cell, a retinal pigment epithelial cell, a hepatic cell, a thyroid cell, a skin cell, a blood cell, a plasma cell, a platelet, a kidney cell, an epithelial cell, a CART cell, an NK cell, and/or a CAR-NK cell, comprises a suicide gene.

In some embodiments, the population of engineered cells causes a reduced amount of or no immune activation upon administration to a recipient subject. In some embodiments, the reduced immune response is compared to the immune response of a patient or control subject administered a population of "wild-type" cells. In some embodiments, the cells cause a reduced amount of systemic TH1 activation or no systemic TH1 activation in the recipient subject. In some embodiments, the cells cause immune activation of reduced amounts of Peripheral Blood Mononuclear Cells (PBMCs) or no PBMCs in the recipient subject. In some embodiments, the cells elicit reduced amounts of donor-specific IgG antibodies or no donor-specific IgG antibodies to the cells upon administration to a recipient subject. In some embodiments, the cells cause a reduced amount of IgM and IgG antibody production or no IgM and IgG antibody production to the cells in the recipient subject. In some embodiments, the cells cause a reduced amount of cytotoxic T cell killing of the cells upon administration to a recipient subject.

1. Therapeutic cells derived from T cells and derived from iPSC

Provided herein are low immunity cells, including, but not limited to, T cells that escape immune recognition. In some embodiments, the low immunity cells are generated (e.g., generated, cultured, or derived) from pluripotent stem cells, such as ipscs, MSCs, and/or ESCs. In some embodiments, the low immunity cells are generated (e.g., produced, cultured, or derived) from T cells, such as primary T cells. In some examples, primary T cells are obtained (e.g., obtained, extracted, removed, or retrieved) from a subject or individual. In some embodiments, primary T cells are generated from a T cell pool such that the T cells are from one or more subjects (e.g., one or more humans including one or more healthy humans). In some embodiments, the T cell pool is from 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., recipient of the administered therapeutic cells). In some embodiments, the T cell pool does not include cells from the patient. In some embodiments, one or more donor subjects from which the T cell pool is obtained are different from the patient.

In some embodiments, the low-immunity cells do not activate the immune response of the patient (e.g., in the recipient of the administration). Provided are methods of treating a disorder comprising repeatedly administering a population of low immunity cells to a subject (e.g., recipient) or patient in need thereof. In some embodiments, a population of low-immunity cells (e.g., low-immunity primary T cells) is administered at least twice (e.g., 2, 3, 4, 5, or more) to a human patient.

In some embodiments, the low-immunity cells do not activate the immune response of the patient (e.g., in the recipient of the administration). Methods are provided for treating a disease by administering a population of low immunity cells to a subject (e.g., recipient) or patient in need thereof. In some embodiments, the low immunity cells described herein comprise T cells engineered (e.g., modified) to express a chimeric antigen receptor, including but not limited to the chimeric antigen receptor described herein. In some examples, the T cells are from a primary T cell population or subpopulation of one or more individuals. In some embodiments, a T cell described herein, such as an engineered or modified T cell, comprises an endogenous T cell receptor that reduces expression.

In some embodiments, the present technology is directed to a low-immunogenicity primary T cell that overexpresses CD47 and CAR, and has reduced or absent expression of MHC class I and/or MHC class II human leukocyte antigens and has reduced or absent expression of TCR complex molecules. The cells outlined herein overexpress CD47 and CAR and escape immune recognition. In some embodiments, the primary T cells display reduced levels or activity of MHC class I antigens, MHC class II antigens, and/or TCR complex molecules. In certain embodiments, the primary T cells overexpress CD47 and CAR and possess genomic modifications at the B2M gene. In some embodiments, the T cell overexpresses CD47 and CAR and possesses a genomic modification at the CIITA gene. In some embodiments, the primary T cells overexpress CD47 and CAR and possess genomic modifications at the TRAC gene. In some embodiments, the primary T cell overexpresses CD47 and CAR and possesses genomic modifications at the TRB gene. In some embodiments, the T cell overexpresses CD47 and CAR and possesses genomic modifications at one or more of the following genes: B2M, CIITA, TRAC and TRB genes.

Exemplary T cells of the disclosure are selected from the group consisting of: cytotoxic T cells, helper T cells, memory T cells, central memory T cells, effector memory RA T cells, regulatory T cells, tissue infiltrating lymphocytes, combinations thereof. In many embodiments, the T cells express CCR7, CD27, CD28, and CD45RA. In some embodiments, the central T cell expresses CCR7, CD27, CD28, and CD45RO. In other embodiments, effector memory T cells express PD1, CD27, CD28, and CD45RO. In other embodiments, effector memory RA T cells express PD1, CD57, and CD45RA.

In some embodiments, the T cell is a modified T cell. In some examples, the modified T cell comprises a modification that results in the cell expressing at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of a damaged cell, a dysplastic cell, an infected cell, an immune cell, an inflammatory cell, a malignant cell, a tissue-deforming cell, a mutant cell, and combinations thereof. In other examples, the modified T cell comprises a modification that results in the cell expressing at least one protein that modulates a biological effect of interest in an adjacent cell, tissue or organ when the cell is in proximity to the adjacent cell, tissue or organ. Modifications useful for primary T cells are described in detail in U.S. Pat. No. 2016/0348073 and WO2020/018620, the disclosures of which are incorporated herein in their entirety.

In some embodiments, the low immunity cells described herein comprise T cells engineered (e.g., modified) to express a chimeric antigen receptor, including but not limited to the chimeric antigen receptor described herein. In some examples, the T cells are a population or subpopulation of primary T cells from one or more individuals. In some embodiments, a T cell described herein, such as an engineered or modified T cell, comprises an endogenous T cell receptor that reduces expression. In some embodiments, T cells described herein, such as engineered or modified T cells, include reduced expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA 4). In other embodiments, a T cell described herein, such as an engineered or modified T cell, comprises reduced expression programmed cell death (PD 1). In certain embodiments, T cells described herein, such as engineered or modified T cells, include CTLA4 and PD1 with reduced expression. In certain embodiments, a T cell described herein, such as an engineered or modified T cell, comprises PD-L1 that enhances expression.

In some embodiments, the low immunity T cell comprises a polynucleotide encoding a CAR, wherein the polynucleotide is inserted at a genomic locus. In some embodiments, the polynucleotide is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, PDGFRa, OLIG2, GFAP, or KDM5D locus. In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene.

2. Chimeric antigen receptor

Provided herein are low immunity cells comprising a Chimeric Antigen Receptor (CAR). In some embodiments, the low immunity cell is a primary T cell or T cell derived from a low immunity pluripotent cell (HIP) provided herein (e.g., a pluripotent stem cell). In some embodiments, the CAR is selected from the group consisting of: first generation CARs, second generation CARs, third generation CARs, and fourth generation CARs.

In some embodiments, the low immunity cells described herein comprise a polynucleotide encoding a Chimeric Antigen Receptor (CAR) (comprising an antigen binding domain). In some embodiments, the low immunity cells described herein comprise a Chimeric Antigen Receptor (CAR) (comprising an antigen binding domain). In some embodiments, the polynucleotide is or comprises a Chimeric Antigen Receptor (CAR) (comprising an antigen binding domain). In some embodiments, the CAR is or comprises a first generation CAR (comprising an antigen binding domain, a transmembrane domain, and at least one signaling domain (e.g., one, two, or three signaling domains)). In some embodiments, the CAR comprises a second generation CAR (comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains). In some embodiments, the CAR comprises a third generation CAR (comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains). In some embodiments, a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene following successful signaling of the CAR. In some embodiments, the antigen binding domain is or comprises an antibody, antibody fragment, scFv, or Fab.

In some embodiments, a low immunity cell described herein (e.g., a low immunity primary T cell or HIP-derived T cell) comprises a polynucleotide encoding a CAR, wherein the polynucleotide is inserted at a genomic locus. In some embodiments, the polynucleotide is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, PDGFRa, OLIG2, GFAP, and/or KDM5D loci. In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene. Any suitable method can be used to insert the CAR into the genomic locus of the low immunity cell, including the gene editing methods described herein (e.g., CRISPR/Cas system).

a) Antigen Binding Domain (ABD) targets antigenic properties of tumor or cancer cells

In some embodiments, the Antigen Binding Domain (ABD) targets an antigenic property of a tumor cell. In other words, the antigen binding domain targets an antigen expressed by a tumor or cancer cell. In some embodiments, ABD binds a tumor-associated antigen. In some embodiments In cases, the antigenic property of the tumor cell (e.g., antigen associated with a tumor or cancer cell) or the tumor-associated antigen is selected from the group consisting of: cell surface receptors, ion channel linked receptors, enzyme linked receptors, G protein coupled receptors, receptor tyrosine kinases, tyrosine kinase related receptors, receptor-like tyrosine phosphatases, receptor serine/threonine kinases, receptor guanylate cyclases (guanyl cyclases), histidine kinase related receptors, epidermal Growth Factor Receptors (EGFR) (including ErbB1/EGFR, erbB2/HER2, erbB3/HER3 and ErbB4/HER 4), fibroblast Growth Factor Receptors (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18 and FGF 21), vascular Endothelial Growth Factor Receptors (VEGFR) (including VEGF-A, VEGF-B, VEGF-C, VEGF-D and PIGF), RET receptors and Eph receptor families (including EphA 1) EphA2, ephA3, ephA4, ephA5, ephA6, ephA7, ephA8, ephA9, ephA10, ephB1, ephB2.EphB3, ephB4, and EphB 6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, spot wilin (bestophin), TMEM16A, GABA receptor, glycine receptor, ABC transporter, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosine (spin) -1-phosphate receptor, transmembrane protein (NMDA-channel), transmembrane protein (S-1-channel; t-cell alpha chain; t-cell beta chain; t-cell gamma chain; t-cell delta chain, CCR7, CD3, CD4, CD5, CD7, CD8, CD11b, CD11c, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1 BB), CD163, F4/80, IL-4Ra, sca-1, CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, NKp46, perforin, CD4+, th1, th2, th17, th40, th22, th9, tfh, treg typically, foxP3+, tr1, th3, treg17, T _RE G. CDCP, NT5E, epCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, PSMA, folate binding proteins, gangliosides (e.g., CD2, CD3, GM 2), lewis-gamma ² 、VEGF、VEGFR1/2/3、αVβ3、α5β1、ErbB1/EGFR, erbB1/HER2, erB3, c-MET, IGF1R, ephA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, tenascin (Tenasmin), PD-1L, BAFF, HDAC, ABL, FLT3, KIT, MET, RET, IL-1β, ALK, RANKL, mTOR, CTLA-4, IL-6R, JAK3, BRAF, PTCH, S Mo Sende (Smoothened), PIGF, ANPEP, TIMP, PLAUR, PTPRJ, LTBR or ANTXR1, folate receptor α (FRa), ERBB2 (HER 2/neu), ephA2, IL-13Ra2, epidermal Growth Factor Receptor (EGFR), mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, MUC16 (CA 125), L1CAM, leY, MSLN, IL Rα1, L1-CAM, tnAg, prostate Specific Membrane Antigen (PSMA), CD1, flT3, FAT 38, TAG 72; CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-11 Ra), PSCA, PRSS21, VEGFR2, lewis Y, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, prostase (Prostase), PAP, ELF2M, ephrinB2, IGF-1 receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, O-acetyl-GD 2, folic acid receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF, CD97, CD179a, ALK, salivary acid, PLACl, globoH, NY-BR 1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, 6K, OR E2, TAESRP 1, NY-1, MAGE-1 Legumain (legumain), HPVE6, E7, ETV6-AML, sperm protein 17, XAGE1, tie2, MAD-CT-1, MAD-CT-2, major histocompatibility Complex class I-related Gene protein (MR 1), urokinase-type cytoplasmic activator receptor (uPAR), fos-related antigen 1, p53 mutant, prostaprotein (prostein), survivin (survivin), telomerase, PCTA-1/Galectin (Galectin) 8, melanA/MART1, ras mutant hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, rhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, enterocarboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, neoantigen, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLa-B, HLA-C, (HLA-A, B, C) CD49f, CD151 CD340, CD200, tkrA, trkB or trkC and/or antigenic fragments or antigenic portions thereof.

b) Antigenic properties of ABD-targeted T cells

In some embodiments, the antigen binding domain targets an antigenic property of a T cell. In some embodiments, the ABD binds an antigen associated with a T cell. In some examples, the antigen is expressed by or located on the surface of a T cell. In some embodiments, the antigenic property of the T cell or T cell-associated antigen is selected from the group consisting of: cell surface receptors for T cells, membrane transport proteins (e.g., active or passive transport proteins such as, for example, ion channel proteins, pore-forming proteins, etc.), transmembrane receptors, membrane enzymes, and/or cell adhesion protein properties. In some embodiments, the antigenic property of the T cell may be a G protein coupled receptor, a receptor tyrosine kinase, a tyrosine kinase related receptor, a receptor-like tyrosine phosphatase, a receptor serine/threonine kinase, a receptor guanylate cyclase, a histidine kinase related receptor, AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD 3 delta); CD 3E (CD 3 epsilon); CD3G (CD 3 γ); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD 3 ζ); CTLA4 (CD 152); ELK1; ERK1 (MAPK 3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2; HLA-DRA; HLA-DRB1; HLA-DRB3; HLA-DRB4; HLA-DRB5; HRAS; IKBKA (CHUK); IKBKB; IKBKE; IKBKG (NEMO); IL2; ITPR1; ITK; JU N; KRAS2; LAT; LCK; MAP2K1 (MEK 1); MAP2K2 (MEK 2); MA P2K3 (MKK 3); MAP2K4 (MKK 4); MAP2K6 (MKK 6); MAP2K7 (MKK 7); MAP3K1 (MEKK 1); MAP3K3; MAP3K4; MAP3K5; MAP3K8; MAP3K14 (NIK); MAPK8 (JNK 1); MAPK9 (JNK 2); MAPK10 (JN K3); MAPK11 (p38β); MAPK12 (p38γ); MAPK13 (p38δ); MAPK14 (p38α); NCK; NFAT1; NFAT2; NFKB1; NFKB2; NFKBIA; NRAS; PAK1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS 34); PIK3CA; PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (perforin); PTEN; RAC1; RAF1; RELA; SDF1; SH P2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1; VAV2; and/or ZAP70.

c) ABD targets antigenic properties of autoimmune or inflammatory disorders

In some embodiments, the antigen binding domain targets an antigenic property of an autoimmune or inflammatory disorder. In some embodiments, ABD binds an antigen associated with an autoimmune or inflammatory disorder. In some examples, the antigen is expressed by a cell associated with an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from: chronic Graft Versus Host Disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, copader's, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, condensed collectin disease, pemphigus vulgaris, grave's disease, autoimmune hemolytic anemia, hemophilia a, primary sjogren's syndrome, thrombotic thrombocytopenia, neuromyelitis optica (neuromyelits optica), evan's syndrome, igM-mediated neuropathy, condensed globulinemia, dermatomyositis, idiopathic thrombocytopenia, joint-adhesive spondylitis, bullous pemphigoid, acquired angioedema, chronic urticaria, antiphospholipid demyelinating multiple neuropathy and autoimmune thrombocytopenia or neutropenia or pure red blood cell dysplasia, while illustrative non-limiting examples of alloimmune diseases include allosensitivity (bliza) (see, e.g., amera, 2015, j. 15, etc.); 931-41) and/or substitution due to hematopoietic or solid organ transplantation (xenostensition), transfusion, pregnancy with fetal allo-sensitivity, neonatal alloimmune thrombocytopenia, hemolytic disease of the neonate, sensitivity to foreign antigens, such as may occur with inherited or acquired deletion disorders treated with enzyme or protein replacement therapies, blood products and/or gene therapies. Allosensitivity, in some examples, refers to development of an immune response (such as circulating antibodies) to human leukocyte antigens that the immune system of the recipient subject or pregnant subject is considered to be non-self antigens. In some embodiments, the antigenic property of the autoimmune or inflammatory disorder is selected from: cell surface receptors, ion channel linked receptors, enzyme linked receptors, G protein coupled receptors, receptor tyrosine kinases, tyrosine kinase related receptors, receptor-like tyrosine phosphatases, receptor serine/threonine kinases, receptor guanylate cyclase (receptor guanylyl cyclase) and/or histidine kinase related receptors.

In some embodiments, the antigen binding domain of the CAR binds to a ligand expressed on B cells, plasma cells, or plasmablasts. In some embodiments, the antigen binding domain of the CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, CD28, TNF, interferon receptor, GM-CSF, ZAP-70, LFA-1, CD3 γ, CD5, or CD2. See, US 2003/0077149; WO 2017/058753; WO 2017/058850, the contents of which are incorporated herein by reference.

d) Antigenic characterization of ABD-targeted senescent cells

In some embodiments, the antigen binding domain targets an antigenic feature of a senescent cell, e.g., urokinase-type cytoplasmic pro-activator receptor (uPAR). In some embodiments, the ABD binds to an antigen associated with a senescent cell. In some examples, the antigen is expressed by senescent cells. In some embodiments, the CAR can be used to treat or prevent a disorder characterized by abnormal accumulation of senescent cells, for example, liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis.

e) Antigenic characterization of ABD-targeted infectious diseases

In some embodiments, the antigen binding domain targets an antigenic feature of an infectious disease. In some embodiments, ABD binds an antigen associated with an infectious disease. In some examples, the antigen is expressed by a cell affected by an infectious disease. In some embodiments, wherein the infectious disease is selected from the group consisting of: HIV, hepatitis B virus, hepatitis C virus, human herpesvirus 8 (HHV-8, kaposi's sarcoma-associated herpesvirus (KSHV)), human T-lymphotropic virus-1 (HTLV-1), merkel cell polyomavirus (MCV), monkey virus 40 (SV 40), epstein-Barr virus, CMV, human papilloma virus. In some embodiments, the antigenic characteristic of the infectious disease is selected from the group consisting of: cell surface receptors, ion channel linked receptors, enzyme linked receptors, G protein coupled receptors, receptor tyrosine kinases, tyrosine kinase related receptors, receptor-like tyrosine phosphatases, receptor serine/threonine kinases, receptor guanylate cyclases, histidine kinase related receptors, HIV Env, gpl20 or CD 4-induced epitopes at HIV-1 Env.

f) ABD binds to cell surface antigens of cells

In some embodiments, the antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, the cell surface antigen is a characteristic of (e.g., expressed by) a particular or specific cell class. In some embodiments, the cell surface antigen is characteristic of more than one cell.

In some embodiments, the a CAR antigen binding domain binds to a cell surface antigen feature of a T cell, such as a cell surface antigen on a T cell. In some embodiments, the antigenic property of the T cell can be a cell surface receptor, a membrane transport protein (e.g., an active or passive transport protein such as, for example, an ion channel protein, a pore forming protein, etc.), a transmembrane receptor, a membrane enzyme, and/or a cell adhesion protein characteristic of the T cell. In some embodiments, the antigenic property of the T cell may be a G protein coupled receptor, a receptor tyrosine kinase, a tyrosine kinase related receptor, a receptor-like tyrosine phosphatase, a receptor serine/threonine kinase, a receptor guanylate cyclase, and/or a histidine kinase related receptor.

In some embodiments, the antigen binding domain of the CAR binds to a T cell receptor. In some embodiments, the T cell receptor may be AKT1; AKT2; AKT3; ATF2; BCL10; CALM1; CD3D (CD 3 delta); CD3E (CD 3 epsilon); CD3G (CD 3 γ); CD4; CD8; CD28; CD45; CD80 (B7-1); CD86 (B7-2); CD247 (CD 3 ζ); CTLA4 (CD 152); ELK1; ERK1 (MAPK 3); ERK2; FOS; FYN; GRAP2 (GADS); GRB2; HLA-DRA; HLA-DRB1; HLA-DRB3; HLA-DRB4; HLA-DRB5; HRAS; IKBKA (CHUK); IKBKB; IKBKE; IKBKG (NEMO); IL2; ITPR1; ITK; JUN; KRAS2; LAT; LCK; MAP2K1 (MEK 1); MAP2K2 (MEK 2); MAP2K3 (MKK 3); MAP2K4 (MKK 4); MAP2K6 (MKK 6); MAP2K7 (MKK 7); MAP3K1 (MEKK 1); MAP3K3; MAP3K4; MAP3K5; MAP3K8; MAP3K14 (NIK); MAPK8 (JNK 1); MAPK9 (JNK 2); MAPK10 (JNK 3); MAPK11 (p38β); MAPK12 (p38γ); MAPK13 (p38δ); MAPK14 (p38α); NCK; NFAT1; NFAT2; NFKB1; NFKB2; NFKBIA; NRAS; PAK1; PAK2; PAK3; PAK4; PIK3C2B; PIK3C3 (VPS 34); PIK3CA; PIK3CB; PIK3CD; PIK3R1; PKCA; PKCB; PKCM; PKCQ; PLCY1; PRF1 (perforin); PTEN; RAC1; RAF1; RELA; SDF1; SHP2; SLP76; SOS; SRC; TBK1; TCRA; TEC; TRAF6; VAV1; VAV2; or ZAP70.

g) Transmembrane domain

In some embodiments, the CAR-transmembrane domain comprises at least the transmembrane region of the α, β or ζ chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof. In some embodiments, the transmembrane domain comprises a transmembrane region of at least CD8 a, CD8 β, 4-1BB/CD137, CD28, CD34, CD4, fceriγ, CD16, OX40/CD134, cd3ζ, cd3ε, cd3γ, cd3δ, tcrα, tcrβ, tcrζ, CD32, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40L/CD154, VEGFR2, FAS, and/or FGFR2B and/or a functional variant thereof.

h) A signaling domain or domains

In some embodiments, a CAR described herein comprises one or at least one signaling domain selected from one or more of the following: B7-1/CD80; B7-2/CD86; B7-H1/PD-L1; B7-H2; B7-H3; B7-H4; B7-H6; B7-H7; BTLA/CD272; CD28; CTLA-4; gi24/VISTA/B7-H5; ICOS/CD278; PD1; PD-L2/B7-DC; PDCD 6); 4-1BB/TNFSF9/CD137;4-1BB ligand/TNFSF 9; BAFF/BLyS/TNFSF13B; BAFF R/TNFRSF13C; CD27/TNFRSF7; CD27 ligand/TNFSF 7; CD30/TNFRSF8; CD30 ligand/TNFSF 8; CD40/TNF RSF5; CD40/TNFSF5; CD40 ligand/TNFSF 5; DR3/TNFRSF25; GIT R/TNFRSF18; GITR ligand/TNFSF 18; HVEM/TNFRSF14; LIGHT/TN FSF14; lymphotoxin-alpha/TNF-beta; OX40/TNFRSF4; OX40 ligand/TNFSF 4; RELT/TNFRSF19L; TACI/TNFRSF13B; TL1A/TNFSF15; TNF-alpha; TNF RII/TNFRSF 1B); 2B4/CD244/SLAMF4; BLAME/SLAMF8; CD2; CD2F-10/SLAMF9; CD48/SLAMF2; CD58/LFA-3; CD84/SLAMF5; CD229/SLAMF3; CRACC/SLAMF7; NTB-A/SLAMF6; SLAM/CD 150); CD2; CD7; CD53; CD82/Kai-1; CD90/Thy1; CD96; CD160; CD200; CD300a/LMIR1; HLA class I; HLA-DR; ikaros; integrin alpha 4/CD49d; integrin alpha 4 beta 1; integrin alpha 4 beta 7/LPAM-1; LAG-3; TCL1A; TCL1B; CRTAM; DAP12; dectin-1/CLEC7A; DPPIV/CD26; eph B6; TIM-1/KIM-1/HAVCR; TIM-4; TSLP; TSLP R; lymphocyte function associated with antigen-1 (LF A-1); NKG2C, CD zeta domain, immune receptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD1, ICOS, antigen-1 (LFA-1) associated lymphocyte function, CD2, CD7, LIGHT, NKG2C, B-H3, ligands that specifically bind to CD83 and/or functional fragments thereof.

In some embodiments, at least one signaling domain comprises a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof. In other embodiments, at least one signaling domain comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In yet other embodiments, at least one signaling domain comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, at least one signaling domain comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes.

In some embodiments, at least two signaling domains comprise a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof. In other embodiments, at least two signaling domains comprise (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In yet other embodiments, at least one signaling domain comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, at least two signaling domains comprise (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes.

In some embodiments, the at least three signaling domains comprise a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof. In other embodiments, at least three signaling domains comprise (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In yet other embodiments, at least three signaling domains comprise (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, the at least three signaling domains comprise (i) a cd3ζ domain or an immunoreceptor tyrosine-based activating motif (ITAM) or functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes.

In some embodiments, at least three signaling domains comprise CD8 a or a functional variant thereof.

In some embodiments, the CAR comprises a cd3ζ domain or an immune receptor tyrosine-based activation motif (ITAM) or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof.

In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof.

In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) A CD28 domain or a 4-1BB domain or a functional variant thereof and/or (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof.

In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes.

i) Domains that induce expression of cytokine genes following successful signaling of CARs

In some embodiments, the first, second, third, or fourth generation CAR further comprises a domain that induces expression of a cytokine gene following successful signaling of the CAR. In some embodiments, the cytokine gene is endogenous or exogenous to the cell of interest, the cell comprising a CAR comprising a domain that induces expression of the cytokine gene following successful signaling of the CAR. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF or IFN-gamma or a functional fragment thereof. In some embodiments, the domain that induces expression of the cytokine gene following successful signaling of the CAR is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments, the domain that induces expression of the cytokine gene following successful signaling of the CAR is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments, the transcription factor or functional domain or fragment thereof is or comprises a nuclear factor of an activated T cell (NFAT), NF-kB, or functional domain or fragment thereof. See, e.g., zhang. C. Et al, engineering CAR-T cells, biomarker research.5:22 (2017); WO 2016126608; sha, H.et al Chimaeric antigen receptor T-cell therapy for tumour immunology.bioscience Reports Jan 27,2017,37 (1).

In some embodiments, the CAR further comprises one or more spacers or hinges, e.g., wherein the spacer is the first spacer between the antigen binding domain and the transmembrane domain. In some embodiments, the first spacer comprises at least a portion of an immunoglobulin constant region or a variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and the signaling domain. In some embodiments, the second spacer is an oligopeptide, for example, wherein the oligopeptide comprises glycine and serine residues, such as, but not limited to, glycine-serine duplex (doubcet). In some embodiments, the CAR comprises two or more spacers, e.g., a spacer between the antigen binding domain and the transmembrane domain and a spacer between the transmembrane domain and the signaling domain. In some embodiments, the spacer is a CD28 hinge, a CD8a hinge, or an IgG4 hinge.

In some embodiments, the CAR further comprises one or more linkers. The format of scFv is generally two variable domains linked by a flexible peptide sequence or "linker" in a position to either VH-linker-VL or VL-linker-VH. Any suitable linker known to those of ordinary skill in the art in view of the description of the invention may be used for the CAR. Examples of suitable linkers include, but are not limited to, GS-based linker sequences and Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 14). In some embodiments, the linker is a GS or gly-ser linker. An exemplary Gly-Ser polypeptide linker comprises the amino acid sequence Ser (Gly ₄ Ser) _n And (Gly) ₄ Ser) _n And/or (Gly) ₄ Ser ₃ ) _n . In some embodiments, n=l. In some embodiments, n=2. In some embodiments, n=3, i.e., ser (Gly ₄ Ser) ₃ . In some embodiments, n=4, i.e., ser (Gly ₄ Ser) ₄ . In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary Gly-Ser polypeptide linker comprises the amino acid sequence Ser (Gly ₄ Ser) _n . In some embodiments, n=l. In some embodiments, n=2. In some embodiments, n=3. In another embodiment, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary Gly-ser polypeptide linker comprises (Gly ₄ Ser) _n . In some embodiments, n=l. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary Gly-ser polypeptide linker comprises (Gly ₃ Ser) _n . In some embodiments, n=l. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6. Another exemplary Gly-ser polypeptide linker comprises (Gly ₄ Ser ₃ ) _n . In some embodiments, n=l. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary Gly-Ser polypeptide linker comprises (Gly 3 Ser) _n . In some embodiments, n=l. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6.

In some embodiments, any of the cells described herein comprise a nucleic acid encoding a CAR or a first generation CAR. In some embodiments, the first generation CAR comprises an antigen binding domain, a transmembrane domain, and a signaling domain. In some embodiments, the signaling domain mediates downstream signaling during T cell activation.

In some embodiments, any of the cells described herein comprise a nucleic acid encoding a CAR or a second generation CAR. In some embodiments, the second generation CAR comprises an antigen binding domain, a transmembrane domain, and two signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a co-stimulatory domain. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation.

In some embodiments, any of the cells described herein comprise a nucleic acid encoding a CAR or a third generation CAR. In some embodiments, a third generation CAR comprises an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a co-stimulatory domain. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and CAR-T cell persistence during T cell activation. In some embodiments, the third generation CAR comprises at least two co-stimulatory domains. In some embodiments, at least two co-stimulatory domains are not identical.

In some embodiments, any of the cells described herein comprise a nucleic acid encoding a CAR or a fourth generation CAR. In some embodiments, the fourth generation CAR comprises an antigen binding domain, a transmembrane domain, and at least two, three, or four signaling domains. In some embodiments, the signaling domain mediates downstream signaling during T cell activation. In some embodiments, the signaling domain is a co-stimulatory domain. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and CAR-T cell persistence during T cell activation.

j) ABD comprising an antibody or antigen binding portion thereof

In some embodiments, the CAR antigen binding domain is or comprises an antibody or antigen binding portion thereof. In some embodiments, the CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, the CAR antigen binding domain comprises an scFv or Fab fragment of a T-cell alpha chain antibody; t-cell beta chain antibodies; t-cell gamma chain antibodies; t-cell delta chain antibodies; CCR7 antibodies; a CD3 antibody; CD4 antibodies; CD5 antibody; a CD7 antibody; CD8 antibodies; CD11b antibodies; CD11c antibody; CD16 antibodies; CD19 antibodies; CD20 antibody; CD21 antibodies; CD22 antibodies; CD25 antibody; CD28 antibody; CD34 antibodies; CD35 antibody; CD40 antibodies; CD45RA antibody; CD45RO antibody; CD52 antibodies; CD56 antibodies; CD62L antibody; CD68 antibody; CD80 antibodies; CD95 antibody; CD117 antibodies; CD127 antibodies; CD133 antibodies; CD137 (4-1 BB) antibody; CD163 antibodies; f4/80 antibody; IL-4Ra antibodies; sca-1 antibody; CTLA-4 antibodies; GITR antibody GARP antibody; LAP antibodies; a granzyme B antibody; LFA-1 antibodies; an MR1 antibody; uPAR antibodies; or transferrin receptor antibodies.

In some embodiments, the CAR comprises a signaling domain that is a co-stimulatory domain. In some embodiments, the CAR comprises a second co-stimulatory domain. In some embodiments, the CAR comprises at least two co-stimulatory domains. In some embodiments, the CAR comprises at least three co-stimulatory domains. In some embodiments, the CAR comprises a co-stimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134/OX40, CD30, CD40, PD1, ICOS, antigen-1 (LFA-1) -associated lymphocyte function, CD2, CD7, LIGHT, NKG2C, B-H3, a ligand that specifically binds to CD 83. In some embodiments, if the CAR comprises two or more co-stimulatory domains, the two co-stimulatory domains are different. In some embodiments, if the CAR comprises two or more co-stimulatory domains, then the two co-stimulatory domains are identical.

In addition to the CARs described herein, various CARs and encoding nucleotide sequences are known in the art and will be suitable for fusion (fusomal) delivery and reprogramming of target cells in vivo and in vitro as described herein. See, for example, WO2013040557; WO2012079000; WO2016030414; smith T, et al, nature nanotechnology.2017.DOI:10.1038/NNANO.2017.57, the disclosure of which is incorporated herein by reference.

3. Therapeutic cells derived from pluripotent stem cells

Provided herein are low immunity cells, including cells derived from pluripotent stem cells that escape immune recognition. In some embodiments, the cells do not activate an immune response in the patient or subject (e.g., in the recipient of the administration). Methods are provided for treating a disorder comprising repeatedly administering a population of low immunity cells to a recipient subject in need thereof.

In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are MHC class I human leukocyte antigens modified to exhibit reduced expression. In other embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are MHC class II human leukocyte antigens modified to exhibit reduced expression. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are MHC class I and class II human leukocyte antigens modified to exhibit reduced expression. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of MHC class I and/or II human leukocyte antigens and exhibit increased CD47 expression. In some examples, the cell overexpresses CD47 by possessing one or more transgenes encoding tolerogenic factors. In some embodiments, the pluripotent stem cells and any cells differentiated from such pluripotent stem cells are modified to exhibit reduced expression of MHC class I and/or II human leukocyte antigens and exhibit increased expression of tolerogenic factors. In some examples, the cell overexpresses CD24 by possessing one or more CD24 transgenes. In some examples, the cells overexpress DUX4 by possessing one or more DUX4 transgenes. The pluripotent stem cells are low-immunity pluripotent cells. The differentiated cells are hypoimmunity cells. Examples of differentiated cells include, but are not limited to, cardiac cells, cardiac progenitor cells, neural cells, glial progenitor cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, chimeric Antigen Receptor (CAR) T cells, NK cells, and/or CAR-NK cells.

Any of the pluripotent stem cells described herein can differentiate into any cell of an organism or tissue. In some embodiments, the cells exhibit reduced expression of MHC class I and/or II human leukocyte antigens. In some examples, MHC class I and/or II human leukocyte antigens are expressed to be reduced compared to unmodified or wild-type cells of the same cell type. In some embodiments, the cells exhibit increased expression of CD 47. In some examples, the expression of CD47 is increased in a cell described herein as compared to an unmodified or wild-type cell of the same cell type. Methods of reducing the amount of MHC class I and/or II human leukocyte antigens and increasing expression of CD47 and one or more tolerogenic factors are described herein.

In some embodiments, the cells used in the methods described herein escape immune recognition and response upon administration to a patient (e.g., recipient subject). Cells can escape killing of immune cells in vitro and in vivo. In some embodiments, the cells escape the killing of macrophages and NK cells. In some embodiments, the cells are ignored by the immune cells or the immune system of the subject. In other words, cells administered according to the methods described herein are not detected by immune cells of the immune system. In some embodiments, the cells are stealth, and thus immune rejection may be avoided.

Methods for determining whether pluripotent stem cells and any cells differentiated from such pluripotent stem cells escape from immune recognition include, but are not limited to, IFN-gamma Elispot assays, microgel cell killing assays, cell implantation animal models, cytokine release assays, ELISA, killing assays using bioluminescence imaging or chromium release assays or xcelligent assays, mixed lymphocyte reactions, immunofluorescence assays, and the like.

The therapeutic cells outlined herein are useful for treating disorders such as, but not limited to, cancer, genetic disorders, chronic infectious diseases, autoimmune disorders, neurological disorders, and the like.

4. Exemplary embodiments of modified cells

In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and decrease expression of MHC class I complexes. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and decrease expression of MHC class II complexes. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and decrease expression of MHC class II and MHC class II complexes.

In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and decreased expression of B2M. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and decreased expression of CIITA. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and decreased expression of NLRC5. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and decrease expression of B2M and CIITA. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and decrease expression of B2M and NLRC5. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and decrease expression of CIITA and NLRC5. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and decrease expression of B2M, CIITA and NLRC5. Any of the cells described herein may also exhibit increased expression of one or more factors selected from the group consisting of, but not limited to: DUX4, CD24, CD27, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8 and Serphinb 9.

In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and at least one other tolerogenic factor, as well as decrease expression of MHC class I complexes. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and at least one other tolerogenic factor, as well as decrease expression of MHC class II complexes. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one other tolerogenic factor, as well as one or more molecules that reduce expression of MHC class II and MHC class II complexes. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one other tolerogenic factor, as well as decreased expression of B2M. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one other tolerogenic factor, as well as decreased expression of CIITA. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one other tolerogenic factor, as well as decreased expression of NLRC5. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one other tolerogenic factor, and one or more molecules B2M and CIITA that reduce expression. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and at least one other tolerogenic factor, and decrease expression of B2M and NLRC5. In some embodiments, the cells and populations thereof exhibit increased expression of CD47 and at least one other tolerogenic factor, and one or more molecules that reduce expression of CIITA and NLRC5. In some embodiments, the cells and populations thereof exhibit one or more molecules that increase expression of CD47 and at least one other tolerogenic factor, and decrease expression of B2M, CIITA and NLRC5. In some embodiments, the tolerogenic agent includes any one selected from the group including, but not limited to: DUX4, CD24, CD27, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8 and Serphinb 9.

Those of ordinary skill in the art will appreciate that the amount of expression, such as a gene, protein, or molecule that increases or decreases expression, can be referenced or compared to a comparable cell. In some embodiments, an engineered stem cell with increased expression of CD47 refers to a modified stem cell with a higher amount of CD47 protein than an unmodified stem cell.

In one embodiment, provided herein are cells (e.g., stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary cells, CAR-T cells, and/or CAR-NK cells) that express exogenous CD47 polypeptides and have reduced expression of one or more MHC class I complex proteins, one or more MHC class II complex proteins, or any combination of MHC class I and class II complex proteins. In another embodiment, the cell expresses an exogenous CD47 polypeptide and expresses a reduced amount of B2M and CIITA polypeptides. In some embodiments, the cells express exogenous CD47 polypeptides and have genetic modifications of B2M and CIITA genes. In some examples, the genetic modification inactivates B2M and CIITA genes.

In some embodiments, the cells (e.g., stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary cells, CAR-T cells, and/or CAR-NK cells) have genetic modifications that inactivate B2M and CIITA genes and express a plurality of exogenous polypeptides selected from the group consisting of: CD47 and DUX4, CD47 and CD24, CD47 and CD27, CD47 and CD46, CD47 and CD55, CD47 and CD59, CD47 and CD200, CD47 and HLA-C, CD and HLA-E, CD47 and HLA-E heavy chain, CD47 and HLA-G, CD and PD-L1, CD47 and IDO1, CD47 and CTLA4-Ig, CD47 and C1-inhibitor, CD47 and IL-10, CD47 and IL-35, CD47 and IL-39, CD47 and FasL, CD47 and CCL21, CD47 and CCL22, CD47 and Mfge8 and CD47 and serpin 9, and any combination thereof. In some examples, the cell also has a genetic modification that inactivates the CD142 gene.

C.CD47

In some embodiments, the disclosure provides cells or populations thereof that have been modified to express a tolerogenic factor (e.g., an immunomodulatory polypeptide) CD47. In some embodiments, the present disclosure provides methods of altering the genome of a cell to express CD47. In some embodiments, the stem cells express exogenous CD47. In some examples, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide. In some embodiments, the cells are genetically modified to comprise an integrated exogenous polynucleotide encoding CD47 using homology directed repair.

CD47 is a leukocyte surface antigen and plays a role in the modulation of cell adhesion and integrins. It is expressed on the cell surface and signals to circulating macrophages that the cells are not to be eaten.

In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide that has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequences listed in NCBI ref. Sequence No. np_001768.1 and np_ 942088.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequences listed in NCBI ref.sequence No. np_001768.1 and np_ 942088.1. In some embodiments, the cell comprises a nucleotide sequence of CD47 that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) with a sequence set forth in NCBI ref.no. nm_001777.3 and nm_ 198793.2. In some embodiments, the cell comprises the nucleotide sequence of CD47, as set forth in NCBI ref. Sequence No. nm_001777.3 and nm_ 198793.2. In some embodiments, the nucleotide sequence encoding the CD47 polynucleotide is a codon optimized sequence. In some embodiments, the nucleotide sequence encoding the CD47 polynucleotide is a human codon optimized sequence.

In some embodiments, the cells comprise a CD47 polypeptide that has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequences listed in NCBI ref. Sequence No. np_001768.1 and np_942088.1. In some embodiments, the cells outlined herein comprise CD47 polypeptides having the amino acid sequences listed in NCBI ref. Sequence No. np_001768.1 and np_942088.1.

Exemplary amino acid sequences of human CD47 with and without signal sequences are provided in table 1.

TABLE 1 amino acid sequence of human CD47

In some embodiments, the cell comprises a CD47 polypeptide that hybridizes to SEQ ID NO:12 (e.g., 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the cell comprises a CD47 polypeptide having the amino acid sequence of SEQ ID NO:12, and a sequence of amino acids. In some embodiments, the cell comprises a CD47 polypeptide that hybridizes to SEQ ID NO:12 (e.g., 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the cell comprises a CD47 polypeptide having the amino acid sequence of SEQ ID NO:12, and a sequence of amino acids.

In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide that hybridizes to SEQ ID NO:13 (e.g., 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide that hybridizes to SEQ ID NO:13 (e.g., 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the nucleotide sequence of SEQ ID NO: 13. In some embodiments, the nucleotide sequence is codon optimized for expression in a particular cell.

In some embodiments, insertion of the polynucleotide encoding CD47 into the genomic locus of the low immunity cell is facilitated using a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein). In some examples, a polynucleotide encoding CD47 is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, PDGFRa, OLIG2, GFAP, or KDM5D locus. In some embodiments, the polynucleotide encoding CD47 is inserted into the B2M gene locus, CIITA gene locus, TRAC gene locus, or TRB gene locus. In some embodiments, the polynucleotide encoding CD47 is inserted into any one of the gene loci described in table 4 provided herein. In certain embodiments, the polynucleotide encoding CD47 is operably linked to a promoter.

In another embodiment, CD47 protein expression is detected using western blot method of cell lysates probed with antibodies to CD47 protein. In another embodiment, the presence of exogenous CD47 mRNA is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

D.CD24

In some embodiments, the disclosure provides cells or populations thereof that have been modified to express a tolerogenic factor (e.g., an immunomodulatory polypeptide) CD24. In some embodiments, the present disclosure provides methods of altering the genome of a cell to express CD24. In some embodiments, the stem cells express exogenous CD24. In some examples, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD24 polypeptide. In some embodiments, the cells are genetically modified to comprise an integrated exogenous polynucleotide encoding CD24 using homology directed repair.

CD24, also known as a thermostable antigen or small cell lung cancer cluster 4 antigen, is a glycosylated phosphatidylinositol-anchored surface protein (Pirruccello et al, J immunol.,1986,136,3779-3784; chen et al, glycobiology,2017,57,800-806). Which binds to Siglec-10 on innate immune cells. CD24 through Siglec-10 has recently been shown to act as an innate immune checkpoint (Barkal et al, nature,2019,572,392-396).

In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide that has at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) with the amino acid sequences listed in NCBI ref.no. np_001278666.1, np_001278667.1, np_001278668.1, and np_ 037362.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having the amino acid sequences listed in NCBI ref.no. np_001278666.1, np_001278667.1, np_001278668.1, and np_ 037362.1.

In some embodiments, the cell comprises a nucleotide sequence that has at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a sequence listed in NCBI ref.no. nm_00129737.1, nm_00129738.1, nm_001291739.1, and nm_ 013230.3. In some embodiments, the cell comprises the nucleotide sequences listed in NCBI ref.no. nm_00129737.1, nm_00129738.1, nm_001291739.1, and nm_ 013230.3.

In another embodiment, CD24 protein expression is detected using western blot method of cell lysates probed with antibodies to CD24 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of exogenous CD24 mRNA.

In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CD24 into a genomic locus of a low immunity cell. In some examples, a polynucleotide encoding CD24 is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, PDGFRa, OLIG2, GFAP, or KDM5D locus. In some embodiments, the polynucleotide encoding CD24 is inserted into the B2M gene locus, CIITA gene locus, TRAC gene locus, or TRB gene locus. In some embodiments, the polynucleotide encoding CD24 is inserted into any one of the gene loci described in table 4 provided herein. In some embodiments, the polynucleotide encoding CD24 is operably linked to a promoter.

E.DUX4

In some embodiments, the disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary cell, or a CAR-T cell), or a population thereof, comprising a genome modified to increase expression of a tolerogenic or immunosuppressive factor, such as DUX 4. In some embodiments, the present disclosure provides methods of altering the genome of a cell to provide increased expression of DUX 4. In one aspect, a cell or population thereof is disclosed that provides for the inclusion of an exogenously expressed DUX4 protein. In some embodiments, the cells are genetically modified to comprise an integrated exogenous polynucleotide encoding DUX4 using homology directed repair. In some embodiments, increasing the expression of DUX4 inhibits, reduces or eliminates the expression of one or more of MHC I molecules-HLA-A, HLA-B, and HLA-C described below.

DUX4 is a transcription factor that is activated in embryonic tissue and induced pluripotent stem cells, and silences in normal, healthy body tissue (Feng et al, 2015,ELife4;De Iaco et al, 2017, nat Genet.,49,941-945; hendrickson et al, 2017, nat Genet.,49,925-934; snider et al, 2010, plos Genet., e1001181; whaddon et al, 2017, nat Genet.). DUX4 expression acts to block IFN- γ mediated induction of Major Histocompatibility Complex (MHC) class I gene expression (e.g., B2M, HLA-A, HLA-B and HLA-C expression). DUX4 expression has been linked to the presentation of inhibited antigen by MHC class I (Chew et al Developmental Cell,2019,50,1-14). DUX4 acts as a transcription factor for gene expression (transcription) procedures during the cleavage phase. Genes of interest include, but are not limited to, coding genes, non-coding genes, and repeat elements.

There are at least two isoforms of DUX4, the longest isoform comprising the DUX 4C-terminal transcriptional activation domain. Isoforms are produced by alternative splicing. See, e.g., geng et al, 2012,Developmental Cell,22,38-51; snider et al 2010, PLoS Genet., e1001181. The active isoforms of DUX4 include their N-terminal DNA binding domain and their C-terminal activation domain. See, e.g., choi et al, 2016,Nucleic Acid Res, 44,5161-5173.

It has been shown that decreasing the number of CpG motifs of DUX4 reduces silencing of the DUX4 transgene (Jagannathan et al, human Molecular Genetics,2016,25 (20): 4419-4431). The nucleic acid sequences provided in Jagannathan et al, supra, represent a sequence of codon changes for DUX4 that includes one or more base substitutions to reduce the total number of CpG sites while preserving the DUX4 protein sequence. Nucleic acid sequences are commercially available from Addgene, catalog number 99281.

In certain aspects, at least one or more polynucleotides can be used to facilitate exogenous expression of DUX4 by a cell, e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary cell, or a CAR-T cell.

In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding DUX4 into a genomic locus of a low immunity cell. In some examples, a polynucleotide encoding DUX4 is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, PDGFRa, OLIG2, GFAP, or KDM5D gene locus. In some embodiments, the polynucleotide encoding DUX4 is inserted into a B2M gene locus, CIITA gene locus, TRAC gene locus, or TRB gene locus. In some embodiments, a polynucleotide encoding DUX4 is inserted into any one of the gene loci described in table 4 provided herein. In certain embodiments, the polynucleotide encoding DUX4 is operably linked to a promoter.

In some embodiments, the polynucleotide sequence encoding DUX4 comprises a polynucleotide sequence comprising a codon-altered nucleotide sequence of DUX4 comprising one or more base substitutions to reduce the total number of CpG sites while preserving the DUX4 protein sequence. In some embodiments, the polynucleotide sequence encoding DUX4 (comprising one or more base substitutions to reduce the total number of CpG sites) hybridizes to SEQ ID NO:1 has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity. In some embodiments, the polynucleotide encoding DUX4 has the sequence of SEQ ID NO:1.

in some embodiments, the polynucleotide sequence encoding DUX4 is a nucleotide sequence encoding a polypeptide sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to a sequence selected from the group consisting of seq id nos: SEQ ID NO as provided in PCT/US 2020/44635: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9.SEQ ID NO: 10. SEQ ID NO: 11. seq id NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO:28 and SEQ ID NO:29. in some embodiments, the polynucleotide sequence encoding DUX4 is a nucleotide sequence encoding a polypeptide sequence selected from the group consisting of: SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9.SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO:28, and SEQ ID NO:29.SEQ ID NO: the amino acid sequences shown in 2 to 29 are shown in figures 1A to 1G of PCT/US 2020/44635.

In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ACN62209.1 or an amino acid sequence set forth in GenBank accession No. ACN 62209.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the sequence set forth in NCBI RefSeq No. np_001280727.1 or the amino acid sequence set forth in NCBI RefSeq No. np_ 001280727.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ACP30489.1 or an amino acid sequence set forth in GenBank accession No. ACP 30489.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in UniProt No. p0cj85.1 or an amino acid sequence set forth in UniProt No. p0cj85.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. AUA60622.1 or an amino acid sequence set forth in GenBank accession No. AUA 60622.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24683.1 or an amino acid sequence set forth in GenBank accession No. ADK 24683.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ACN62210.1 or an amino acid sequence set forth in GenBank accession No. ACN 62210.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24706.1 or an amino acid sequence set forth in GenBank accession No. ADK 24706.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24685.1 or an amino acid sequence set forth in GenBank accession No. ADK 24685.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ACP30488.1 or an amino acid sequence set forth in GenBank accession No. ACP 30488.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24687.1 or an amino acid sequence set forth in GenBank accession No. ADK 24687.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ACP30487.1 or an amino acid sequence set forth in GenBank accession No. ACP 30487.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24717.1 or an amino acid sequence set forth in GenBank accession No. ADK 24717.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24690.1 or an amino acid sequence set forth in GenBank accession No. ADK 24690.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the sequence listed in GenBank accession No. ADK24689.1 or the amino acid sequence listed in GenBank accession No. ADK 24689.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24692.1 or an amino acid sequence set forth in GenBank accession No. ADK 24692.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24693.1 or an amino acid sequence set forth in GenBank accession No. ADK 24693.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24712.1 or an amino acid sequence set forth in GenBank accession No. ADK 24712.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24691.1 or an amino acid sequence set forth in GenBank accession No. ADK 24691.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in UniProt No. p0cj87.1 or an amino acid sequence set forth in UniProt No. p0cj87.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24714.1 or an amino acid sequence set forth in GenBank accession No. ADK 24714.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24684.1 or an amino acid sequence set forth in GenBank accession No. ADK 24684.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24695.1 or an amino acid sequence set forth in GenBank accession No. ADK 24695.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to a sequence set forth in GenBank accession No. ADK24699.1 or an amino acid sequence set forth in GenBank accession No. ADK 24699.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the sequence set forth in NCBI RefSeq No. np_001768.1 or the amino acid sequence set forth in NCBI RefSeq No. np_001768. In some examples, the DUX4 polypeptide comprises an amino acid sequence that has at least 95% sequence identity to the sequence listed in NCBI refseqno. np_942088.1 or the amino acid sequence listed in NCBI RefSeq No. np_ 942088.1. In some examples, the DUX4 polypeptide comprises an amino acid sequence that hybridizes to the sequence set forth in SEQ ID NO:28 or PCT/US2020/44635, SEQ ID NO:28 has at least 95% sequence identity to the amino acid sequence of seq id no. In some examples, the DUX4 polypeptide comprises an amino acid sequence that hybridizes to the sequence set forth in SEQ ID NO:29 or PCT/US2020/44635, SEQ ID NO:29 has at least 95% sequence identity to the amino acid sequence of seq id no.

In other embodiments, expression vectors are used to assist in expression of tolerogenic factors. In some embodiments, the expression vector comprises a sequence in which the polynucleotide sequence encoding DUX4 is codon altered, comprising one or more base substitutions to reduce the total number of CpG sites while preserving the DUX4 protein sequence. In some examples, the sequence of codon changes for DUX4 comprises the sequence of SEQ ID NO:1. in some examples, the sequence of the codon change of DUX4 is the SEQ ID NO of PCT/US 2020/44635: 1. in other embodiments, the expression vector comprises a polynucleotide sequence encoding DUX4 comprising the sequence of SEQ ID NO:1. in some embodiments, the expression vector comprises a polynucleotide sequence encoding a DUX4 polypeptide sequence having at least 95% sequence identity to a sequence selected from the group consisting of seq id nos: SEQ ID NO of PCT/US 2020/44635: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO:28 and SEQ ID NO:29. in some embodiments, the expression vector comprises a polynucleotide sequence encoding a DUX4 polypeptide sequence selected from the group consisting of: SEQ ID NO of PCT/US 2020/44635: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. SEQ ID NO: 18. SEQ ID NO: 19. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO:28 and SEQ ID NO:29.

The increase in DUX4 expression may be assayed using known techniques, such as western blot methods, ELISA assays, FACS assays, immunoassays, and the like.

F.CIITA

In certain aspects, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of MHC class II genes by targeting and modulating (e.g., reducing or eliminating) expression of a class II transductor (CIITA). In some embodiments, modulation occurs using a gene editing (e.g., CRISPR/Cas) system. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, a DNA-based method selected from the group consisting of: knocking out or attenuating using a method selected from the group consisting of: CRISPR, TALEN, zinc finger nucleases, homing endonucleases and meganucleases are modulated. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, an RNA-based method selected from the group consisting of: shRNA, siRNA, miRNA and CRISPR interference (CRISPRi) are modulated. In some embodiments, modulation of CIITA expression includes, but is not limited to, reduced transcription, reduced mRNA stability (such as through RNAi machinery), and reduced protein mass.

CIITA is a member of the LR or Nucleotide Binding Domain (NBD) leucine-rich repeat (LRR) family of proteins and regulates MHC II transcription by association with MHC enhancers.

In some embodiments, the polynucleotide sequence of interest is a variant of CIITA. In some embodiments, the polynucleotide sequence of interest is a homolog of CIITA. In some embodiments, the polynucleotide sequence of interest is a heterologous homolog of CIITA.

In some embodiments, reduced or eliminated expression CIITA reduces or eliminates expression of one or more of MHC class II described below, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

In some embodiments, the cells outlined herein comprise a genetic modification that targets the CIITA gene. In some embodiments, the genetic modification of the CIITA gene by rare-cutting endonucleases comprises a protein or polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence that specifically targets the CIITA gene. In some embodiments, the at least one guide ribonucleic acid sequence that specifically targets the CIITA gene is selected from the group consisting of: WO2016183041 annex 1 or SEQ ID NO:5184 to 36352, the disclosures of which are incorporated by reference in their entirety. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted into the CIITA gene.

Assays for testing whether the CIITA gene has been inactivated are known and described herein. In one embodiment, the genetic modification of the CIITA gene and the reduction in HLA-II expression by PCR can be assayed by FACS analysis. In another embodiment, CIITA protein expression is detected using western blot method of cell lysates probed with antibodies to CIITA proteins. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactive genetic modifications.

G.B2M

In certain embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) the expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) the expression of accessory chain B2M. In some embodiments, modulation occurs using a gene editing (e.g., CRISPR/Cas) system. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, a DNA-based method selected from the group consisting of: knocking out or attenuating using a method selected from the group consisting of: CRISPR, TALEN, zinc finger nucleases, homing endonucleases and meganucleases are modulated. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, an RNA-based method selected from the group consisting of: shRNA, siRNA, miRNA and CRISPR interference (CRISPRi) are modulated. In some embodiments, modulation of B2M expression includes, but is not limited to, reduced transcription, reduced mRNA stability (such as through RNAi machinery), and reduced protein mass.

By modulating (e.g., reducing or deleting) the expression of B2M, surface transport of MHC-I molecules is blocked and such cells exhibit immune tolerance when implanted into a recipient subject. In some embodiments, the cells are considered hypoimmunity after administration, e.g., in a recipient subject or patient.

In some embodiments, the polynucleotide sequence of interest provided herein is a variant of B2M. In some embodiments, the polynucleotide sequence of interest is a homolog of B2M. In some embodiments, the polynucleotide sequence of interest is a B2M heterologous homolog.

In some embodiments, the reduced or eliminated expression of B2M reduces or eliminates expression of one or more of MHC I molecules-HLA-A, HLA-B, and HLA-C described below.

In some embodiments, the low immunity cells outlined herein comprise a genetic modification that targets the B2M gene. In some embodiments, the genetic modification of the B2M gene by rare-cutting endonuclease targeting comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence that specifically targets the B2M gene. In some embodiments, the at least one guide ribonucleic acid sequence that specifically targets the B2M gene is selected from the group consisting of: appendix 2 of WO2016/183041 or SEQ ID NO:81240 to 85644, the disclosures of which are incorporated by reference in their entirety. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., chimeric antigen receptor, CD47, or another tolerogenic agent disclosed herein) is inserted into the B2M gene.

Assays to test whether the B2M gene has been inactivated are known and described herein. In one embodiment, the genetic modification of the B2M gene and the reduction in HLA-I expression by PCR can be assayed by FACS analysis. In another embodiment, B2M protein expression is detected using western blot method of cell lysates probed with antibodies to B2M protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactive genetic modifications.

H.NLRC5

In certain aspects, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting and modulating (e.g., reducing or eliminating) expression of the NLR family, CARD domain comprising 5/NOD27/CLR16.1 (NLRC 5). In some embodiments, modulation occurs using a gene editing (e.g., CRISPR/Cas) system. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, a DNA-based method selected from the group consisting of: knocking out or attenuating using a method selected from the group consisting of: CRISPR, TALEN, zinc finger nucleases, homing endonucleases and meganucleases are modulated. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, an RNA-based method selected from the group consisting of: shRNA, siRNA, miRNA and CRISPR interference (CRISPRi) are modulated. In some embodiments, modulation of NLRC5 expression includes, but is not limited to, reduced transcription, reduced mRNA stability (such as through RNAi machinery), and reduced protein mass.

NLRC5 is a regulator of MHC-I mediated immune responses, and like CIITA, NLRC5 is highly inducible by IFN-gamma and can translocate into the nucleus. NLRC5 activates the promoter of the MHC-I gene and induces transcription of MHC-I and related genes involved in MHC-I antigen presentation.

In some embodiments, the polynucleotide sequence of interest is a variant of NLRC 5. In some embodiments, the polynucleotide sequence of interest is a homolog of NLRC 5. In some embodiments, the polynucleotide sequence of interest is a heterologous homolog of NLRC 5.

In some embodiments, reduced or eliminated expression of NLRC5 reduces or eliminates expression of one or more of MHC I molecules-HLA-A, HLA-B, and HLA-C described below.

In some embodiments, the cells outlined herein comprise a genetic modification that targets the NLRC5 gene. In some embodiments, the genetic modification that targets the NLRC5 gene by rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence that specifically targets the NLRC5 gene. In some embodiments, the at least one guide ribonucleic acid sequence that specifically targets the NLRC5 gene is selected from the group consisting of: accessory 3 of WO2016183041 or SEQ ID NO of Table 14: 36353 to 81239, the disclosures of which are incorporated by reference in their entirety.

Assays to test whether the NLRC5 gene has been inactivated are known and described herein. In one embodiment, the modification of the NLRC5 gene and the reduction of HLA-I expression by PCR can be assayed by FACS analysis. In another embodiment, NLRC5 protein expression is detected using western blot method of cell lysate probed with an antibody to NLRC5 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactive genetic modifications.

I.TRAC

In many embodiments, the technology disclosed herein adjustably modulates (e.g., reduces or eliminates) the expression of TCR genes (including TRAC genes) by adjustably targeting and modulating (e.g., reducing or eliminating) the expression of T cell receptor alpha chain constant regions. In some embodiments, modulation occurs using a gene editing (e.g., CRISPR/Cas) system. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, the modulation occurs using a DNA-based method selected from the group consisting of: knocking out or attenuating using a method selected from the group consisting of: CRISPR, TALEN, zinc finger nucleases, homing endonucleases and meganucleases. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, an RNA-based method selected from the group consisting of: shRNA, siRNA, miRNA and CRISPR interference (CRISPRi) are modulated. In some embodiments, modulation of TRAC expression includes, but is not limited to, reduced transcription, reduced mRNA stability (such as through RNAi machinery), and reduced protein mass.

By modulating (e.g., reducing or deleting) the expression of TRAC, surface transport of TCR molecules is blocked. In some embodiments, the cell also has a reduced ability to induce an immune response in the recipient subject.

In some embodiments, the polynucleotide sequence of interest of the present technology is a variant of TRAC. In some embodiments, the polynucleotide sequence of interest is a homolog of TRAC. In some embodiments, the polynucleotide sequence of interest is a heterologous homolog of TRAC.

In some embodiments, reduced or eliminated expression of TRAC reduces or eliminates TCR surface expression.

In some embodiments, cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, comprise a regulatable genetic modification at a gene locus encoding a TRAC protein. In other words, the cell comprises a regulatable genetic modification at the TRAC locus. In some examples, the nucleotide sequence encoding the TRAC protein is listed in Genbank No. X02592.1. In some examples, the TRAC Gene locus is described in RefSeq.No. NG_001332.3 and NCBI Gene ID No.28755. In some examples, the amino acid sequence of TRAC is described as Uniprot No. P01848. Additional descriptions of TRAC proteins and gene loci can be found in Uniprot No. P01848, HGNC Ref.No.12029 and OMIM Ref.No. 186880.

In some embodiments, the low immunity cells outlined herein comprise a regulatable genetic modification that targets the TRAC gene. In some embodiments, the adjustable genetic modification targeting the TRAC gene comprises an adjustable Cas protein or an adjustable polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence specifically targeting the TRAC gene by an adjustable rare-cutting endonuclease. In some embodiments, the at least one leader ribonucleic acid sequence that specifically targets the TRAC gene is selected from the group consisting of: SEQ ID NO of US 20160348073: 532 to 609 and 9102 to 9797, which are incorporated herein by reference.

Assays to test whether the TRAC gene has been inactivated are known and described herein. In one embodiment, the modification of the TRAC gene and the reduction of TCR expression by PCR can be assayed by FACS analysis. In another embodiment, TRAC protein expression is detected using the western blot method of cell lysates probed with antibodies to TRAC protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactive genetic modifications.

In some embodiments, the low immunity cells outlined herein comprise a regulatable knockdown of TRAC expression such that the cells are regulatable TRAC ^-/- . In some embodiments, the low immunity cells outlined herein adjustably introduce indels into the TRAC gene loci such that the cells are adjustably TRAC ^indel/indel . In some embodiments, the low immunity cells outlined herein comprise a regulatable decrease in TRAC expression such that the cells are regulatable TRAC ^knock ^down 。

J.TRB

In many embodiments, the technology disclosed herein adjustably modulates (e.g., reduces or eliminates) the expression of TCR genes, including genes encoding T cell antigen receptors, beta chains (e.g., TRB, TRBC, or TCRB genes), by adjustably targeting and modulating (e.g., reducing or eliminating) the expression of T cell receptor beta chain constant regions. In some embodiments, modulation occurs using a gene editing (e.g., CRISPR/Cas) system. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, the modulation occurs using a DNA-based method selected from the group consisting of: knocking out or attenuating using a method selected from the group consisting of: CRISPR, TALEN, zinc finger nucleases, homing endonucleases and meganucleases. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, an RNA-based method selected from the group consisting of: shRNA, siRNA, miRNA and CRISPR interference (CRISPRi) are modulated. In some embodiments, modulation of TRB expression includes, but is not limited to, reduced transcription, reduced mRNA stability (such as through RNAi machinery), and reduced protein mass.

By modulating (e.g., reducing or deleting) the expression of TRB, surface transport of TCR molecules is blocked. In some embodiments, the cell also has a reduced ability to induce an immune response in the recipient subject.

In some embodiments, the polynucleotide sequence of interest of the present technology is a variant of TRB. In some embodiments, the polynucleotide sequence of interest is a homolog of TRB. In some embodiments, the polynucleotide sequence of interest is a heterologous homolog of TRB.

In some embodiments, reduced or eliminated expression of TRB reduces or eliminates TCR surface expression.

In some embodiments, cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, comprise a regulatable genetic modification at a genetic locus encoding a TRB protein. In other words, the cell comprises a regulatable genetic modification at the TRB gene locus. In some examples, the nucleotide sequence encoding the TRB protein is listed in UniProt No. p0dse2. In some examples, the TRB Gene locus is described in refseq.no. ng_001333.2 and NCBI Gene ID No.6957. In some examples, the amino acid sequence of TRB is described as Uniprot No. p01848. Additional descriptions of TRB proteins and gene loci can be found in GenBank No. l36092.2, uniprot No. p0dse2, and HGNC ref No. 12155.

In some embodiments, the low immunity cells outlined herein comprise a regulatable genetic modification that targets a TRB gene. In some embodiments, the adjustable genetic modification of the targeted TRB gene comprises an adjustable Cas protein or an adjustable polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence of the specific targeted TRB gene by an adjustable rare-cutting endonuclease. In some embodiments, the at least one leader ribonucleic acid sequence that specifically targets the TRB gene is selected from the group consisting of: SEQ ID NO of US 20160348073: 610-765 and 9798-10532, which are incorporated herein by reference.

Assays for testing whether the TRB gene has been inactivated are known and described herein. In one embodiment, the genetic modification of the TRB gene and the reduction in TCR expression by PCR can be assayed by FACS analysis. In another embodiment, TRB protein expression is detected using western blot method of cell lysates probed with antibodies to TRB protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactive genetic modifications.

In some embodiments, the low immunity cells outlined herein comprise a regulatable knockdown of TRB expression such that the cells are regulatable TRB ^-/- . In some embodiments, a low immunity cell as outlined herein adjustably introduces an indel to a TRB gene locus such that the cell is adjustably TRB ^indel/indel . In some embodiments, the low immunity cells outlined herein comprise a modulated attenuation of TRB expression such that the cells are regulatably TRB ^knock ^down 。

K.CD142

In certain aspects, the techniques disclosed herein modulate (e.g., reduce or eliminate) the expression of CD142 (also known as tissue factor, factor III, and F3). In some embodiments, modulation occurs using a gene editing (e.g., CRISPR/Cas) system. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, the modulation occurs using a DNA-based method selected from the group consisting of: knocking out or attenuating using a method selected from the group consisting of: CRISPR, TALEN, zinc finger nucleases, homing endonucleases and meganucleases. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, an RNA-based method selected from the group consisting of: shRNA, siRNA, miRNA and CRISPR interference (CRISPRi) are modulated. In some embodiments, modulation of CD142 expression includes, but is not limited to, reduced transcription, reduced mRNA stability (such as through RNAi machinery), and reduced protein mass.

In some embodiments, the polynucleotide sequence of interest is CD142 or a variant of CD 142. In some embodiments, the polynucleotide sequence of interest is a homolog of CD 142. In some embodiments, the polynucleotide sequence of interest is a heterologous homolog of CD 142.

In some embodiments, the cells outlined herein comprise a genetic modification that targets the CD142 gene. In some embodiments, the genetic modification that targets the CD142 gene by rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid (gRNA) sequence that specifically targets the CD142 gene. Useful methods of identification are described below for the gRNA sequence of target CD 142.

Assays for testing whether the CD142 gene has been inactivated are known and described herein. In one embodiment, the genetic modification of the CD142 gene and the reduction in CD142 expression by PCR can be assayed by FACS analysis. In another embodiment, CD142 protein expression is detected using western blot method of cell lysates probed with antibodies to CD142 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactive genetic modifications.

Useful genomic, polynucleotide, and polypeptide information about human CD142 is provided, for example, in GeneCard Identifier GC M094530, HGNC No.3541, NCBI Gene ID 2152, NCBI RefSeq nos. nm_001178096.1, nm_001993.4, np_001171567.1 and np_001984.1, uniProt No. p13726, and the like.

L.CTLA4

In certain aspects, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of CTLA 4. In some embodiments, modulation occurs using a gene editing (e.g., CRISPR/Cas) system. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, the modulation occurs using a DNA-based method selected from the group consisting of: knocking out or attenuating using a method selected from the group consisting of: CRISPR, TALEN, zinc finger nucleases, homing endonucleases and meganucleases. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, an RNA-based method selected from the group consisting of: shRNA, siRNA, miRNA and CRISPR interference (CRISPRi) are modulated. In some embodiments, modulation of CTLA4 expression includes, but is not limited to, reduced transcription, reduced mRNA stability (such as through RNAi machinery), and reduced protein mass.

In some embodiments, the polynucleotide sequence of interest is CTLA4 or a variant of CTLA 4. In some embodiments, the polynucleotide sequence of interest is a homolog of CTLA 4. In some embodiments, the polynucleotide sequence of interest is a heterohomolog of CTLA 4.

In some embodiments, the cells outlined herein comprise a genetic modification that targets a CTLA4 gene. In certain embodiments, the primary T cells comprise a genetic modification that targets a CTLA4 gene. Genetic modification can reduce expression of CTLA4 polynucleotides and CTLA4 polypeptides in T cells (including primary T cells and CAR-T cells). In some embodiments, the genetic modification that targets the CTLA4 gene by rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid (gRNA) sequence that specifically targets the CTLA4 gene. Useful methods for identifying gRNA sequences for CTLA4 of interest are described below.

Assays for testing whether CTLA4 genes have been inactivated are known and described herein. In one embodiment, the modification of CTLA4 gene and reduction of CTLA4 expression by PCR can be assayed by FACS analysis. In another embodiment, CTLA4 protein expression is detected using western blot method of cell lysates probed with antibodies to CTLA4 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactive genetic modifications.

Useful genomic, polynucleotide, and polypeptide information about human CTLA4 is provided, for example, in GeneCard Identifier GC02P203867, HGNC No.2505, NCBI Gene ID 1493, NCBI RefSeq No. nm_005214.4, nm_001037631.2, np_001032720.1 and np_005205.2, uniProt No. P16410, and the like.

M.PD1

In certain aspects, the techniques disclosed herein modulate (e.g., reduce or eliminate) expression of PD 1. In some embodiments, modulation occurs using a gene editing (e.g., CRISPR/Cas) system. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, the modulation occurs using a DNA-based method selected from the group consisting of: knocking out or attenuating using a method selected from the group consisting of: CRISPR, TALEN, zinc finger nucleases, homing endonucleases and meganucleases. In some embodiments, the modification is transient (including, for example, by using an siRNA approach). In some embodiments, an RNA-based method selected from the group consisting of: shRNA, siRNA, miRNA and CRISPR interference (CRISPRi) are modulated. In some embodiments, modulation of PD1 expression includes, but is not limited to, reduced transcription, reduced mRNA stability (such as through RNAi machinery), and reduced protein mass.

In some embodiments, the polynucleotide sequence of interest is PD1 or a variant of PD 1. In some embodiments, the polynucleotide sequence of interest is a homolog of PD 1. In some embodiments, the polynucleotide sequence of interest is a heterologous homolog of PD 1.

In some embodiments, the cells outlined herein comprise a genetic modification that targets a gene encoding a programmed cell death protein 1 (PD 1) protein or a PDCD1 gene. In certain embodiments, the primary T cells comprise a genetic modification that targets the PDCD1 gene. Genetic modification can reduce expression of PD1 polynucleotides as well as PD1 polypeptides in T cells (including primary T cells and CAR-T cells). In some embodiments, the genetic modification that targets the PDCD1 gene by rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid (gRNA) sequence that specifically targets the PDCD1 gene. Useful methods for identifying the gRNA sequence for target PD1 are described below.

Assays for testing whether the PDCD1 gene has been inactivated are known and described herein. In one embodiment, the gene modification of the PDCD1 gene and the reduction in PD1 expression by PCR can be assayed by FACS analysis. In another embodiment, PD1 protein expression is detected using western blot method of cell lysates probed with antibodies to PD1 protein. In another embodiment, reverse transcriptase polymerase chain reaction (RT-PCR) is used to confirm the presence of inactive genetic modifications.

Useful genomic, polynucleotide and polypeptide information about human PD1 (including PDCD1 Gene) is provided, for example, in GeneCard Identifier GC M241849, HGNC No.8760, NCBI Gene ID 5133, uniprot No. q15116, and NCBI RefSeq No. nm_005018.2 and np_005009.2.

N. additional tolerogenic factors

In certain embodiments, one or more tolerogenic factors can be inserted or reinserted into the genome-edited cell to create an immune-privileged universal donor cell, such as a universal donor stem cell, a universal donor T cell, or a universal donor cell. In certain embodiments, the hypoimmunity cells disclosed herein have been further modified to express one or more tolerogenic factors.

Exemplary tolerogenic factors include, without limitation, CD47, DUX4, CD24, CD27, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8, serphinb 9, CD16 Fc receptor, IL15-RF, CD16, CD52, H2-M3 and CD35. In some embodiments, the tolerogenic agent is selected from the group consisting of: CD200, HLA-G, HLA-E, HLA-C, HLA-E heavy chain, PD-L1, IDO1, CTLA4-Ig, IL-10, IL-35, fasL, serpinb9, CCL21, CCL22 and Mfge8. In some embodiments, the tolerogenic agent is selected from the group consisting of: DUX4, HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor and IL-35. In some embodiments, the tolerogenic agent is selected from the group consisting of: HLA-C, HLA-E, HLA-F, HLA-G, PD-L1, CTLA-4-Ig, C1-inhibitor and IL-35.

In some examples, a gene editing system, such as a CRISPR/Cas system, is used to facilitate insertion of a tolerogenic factor, such as a tolerogenic factor, to a safe harbor or target locus, such as an AAVS1 locus, to actively suppress immune rejection. In some examples, the tolerance cause is inserted into the safe harbor or target locus using an expression vector. In some embodiments, the safe harbor or target locus is an AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, PDGFRa, OLIG2, GFAP, or KDM5D locus.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low-immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express CD 47. In some embodiments, the present disclosure provides methods of altering the genome of a cell to express CD 47. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of CD47 into a cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: SEQ ID NO of Table 29 of WO 2016183041: 200784 to 231885, which are incorporated herein by reference. In some embodiments, the primary cells include, but are not limited to, heart cells, heart progenitor cells, nerve cells, glial progenitor cells, endothelial cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, T cells, B cells, or NK cells. In some embodiments, stem cells include, but are not limited to, embryonic stem cells, induced stem cells, mesenchymal stem cells, and hematopoietic stem cells.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low-immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express HLA-C. In some embodiments, the present disclosure provides methods of altering the genome of a cell to express HLA-C. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of HLA-C into a cell strain. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: SEQ ID NO of Table 10 of WO 2016183041: 3278 to 5183, which are incorporated herein by reference. In some embodiments, the primary cells include, but are not limited to, heart cells, heart progenitor cells, nerve cells, glial progenitor cells, endothelial cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, T cells, B cells, or NK cells. In some embodiments, stem cells include, but are not limited to, embryonic stem cells, induced stem cells, mesenchymal stem cells, and hematopoietic stem cells.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low-immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express HLA-E. In some embodiments, the disclosure provides methods of altering the genome of a cell to express HLA-E. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of HLA-E into a cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: SEQ ID NO of Table 19 of WO 2016183041: 189859 to 193183, which are incorporated herein by reference. In some embodiments, the primary cells include, but are not limited to, heart cells, heart progenitor cells, nerve cells, glial progenitor cells, endothelial cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, T cells, B cells, or NK cells. In some embodiments, stem cells include, but are not limited to, embryonic stem cells, induced stem cells, mesenchymal stem cells, and hematopoietic stem cells.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low-immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express HLA-F. In some embodiments, the disclosure provides methods of altering the genome of a cell to express HLA-F. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of HLA-F into a cell strain. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: SEQ ID NO of Table 45 of WO 2016183041: 688808 to 399754, which are incorporated herein by reference. In some embodiments, the primary cells include, but are not limited to, heart cells, heart progenitor cells, nerve cells, glial progenitor cells, endothelial cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, T cells, B cells, or NK cells. In some embodiments, stem cells include, but are not limited to, embryonic stem cells, induced stem cells, mesenchymal stem cells, and hematopoietic stem cells.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low-immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express HLA-G. In some embodiments, the disclosure provides methods of altering the genome of a cell to express HLA-G. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of HLA-G into a stem cell line. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: SEQ ID NO of Table 18 of WO 2016183041: 188372 to 189858, which are incorporated herein by reference. In some embodiments, the primary cells include, but are not limited to, heart cells, heart progenitor cells, nerve cells, glial progenitor cells, endothelial cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, T cells, B cells, or NK cells. In some embodiments, stem cells include, but are not limited to, embryonic stem cells, induced stem cells, mesenchymal stem cells, and hematopoietic stem cells.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low-immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express PD-L1. In some embodiments, the disclosure provides methods of altering the genome of a cell to express PD-L1. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of PD-L1 into a stem cell strain. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: SEQ ID NO of Table 21 of WO 2016183041: 193184 to 200783, which are incorporated herein by reference. In some embodiments, the primary cells include, but are not limited to, heart cells, heart progenitor cells, nerve cells, glial progenitor cells, endothelial cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, T cells, B cells, or NK cells. In some embodiments, stem cells include, but are not limited to, embryonic stem cells, induced stem cells, mesenchymal stem cells, and hematopoietic stem cells.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express CTLA 4-Ig. In some embodiments, the disclosure provides methods of altering the genome of a cell to express CTLA 4-Ig. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of CTLA4-Ig into a stem cell strain. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: WO2016183041, including any of the ones disclosed in the sequence listing.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low-immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express a CI-inhibitor. In some embodiments, the present disclosure provides methods of altering the genome of a cell to express a CI-inhibitor. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids may be used to facilitate insertion of the CI-inhibitor into the stem cell strain. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: WO2016183041, including any of the ones disclosed in the sequence listing. In some embodiments, the primary cells include, but are not limited to, heart cells, heart progenitor cells, nerve cells, glial progenitor cells, endothelial cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, T cells, B cells, or NK cells. In some embodiments, stem cells include, but are not limited to, embryonic stem cells, induced stem cells, mesenchymal stem cells, and hematopoietic stem cells.

In some embodiments, the disclosure provides cells (e.g., primary cells and/or low-immunity stem cells and derivatives thereof) or populations thereof, comprising a genome in which the cell genome has been modified to express IL-35. In some embodiments, the disclosure provides methods of altering the genome of a cell to express IL-35. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of IL-35 into a stem cell strain. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: WO2016183041, including any of the ones disclosed in the sequence listing. In some embodiments, the primary cells include, but are not limited to, heart cells, heart progenitor cells, nerve cells, glial progenitor cells, endothelial cells, pancreatic islet cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, T cells, B cells, or NK cells. In some embodiments, stem cells include, but are not limited to, embryonic stem cells, induced stem cells, mesenchymal stem cells, and hematopoietic stem cells.

In some embodiments, the tolerogenic factors are expressed in the cells using an expression vector. For example, an expression vector for expressing CD47 in a cell comprises a polynucleotide sequence encoding CD 47. The expression vector may be an inducible expression vector. The expression vector may be a viral vector, such as, but not limited to, a lentiviral vector.

In some embodiments, the disclosure provides a cell (e.g., a primary cell and/or a low immunity stem cell and derivatives thereof) or population thereof, comprising a genome in which the cell genome has been modified to express any one of the polypeptides selected from the group consisting of: HLA-A, HLA-B, HLA-C, RFX-ANK, CIITA, NFY-A, NLRC5, B2M, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAP1, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS and IDO1. In some embodiments, the present disclosure provides methods of altering the genome of a cell to express any one of the polypeptides selected from the group consisting of: HLA-A, HLA-B, HLA-C, RFX-ANK, CIITA, NFY-A, NLRC5, B2M, RFX5, RFX-AP, HLA-G, HLA-E, NFY-B, PD-L1, NFY-C, IRF1, TAP1, GITR, 4-1BB, CD28, B7-1, CD47, B7-2, OX40, CD27, HVEM, SLAM, CD226, ICOS, LAG3, TIGIT, TIM3, CD160, BTLA, CD244, LFA-1, ST2, HLA-F, CD30, B7-H3, VISTA, TLT, PD-L2, CD58, CD2, HELIOS and IDO1. In certain aspects, at least one ribonucleic acid or at least one pair of ribonucleic acids can be used to facilitate insertion of the selected polypeptide into a stem cell strain. In certain embodiments, at least one ribonucleic acid or at least one pair of ribonucleic acids is selected from the group consisting of: any of the attachments 1 to 47 and the disclosures in the sequence listing of WO2016183041, the disclosures of which are incorporated herein by reference.

In some embodiments, a suitable gene editing system (e.g., CRISPR/Cas system or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding a tolerogenic factor into a genomic locus of a low-immunity cell. In some examples, a polynucleotide encoding a tolerogenic factor is inserted into a safe harbor or target locus, such as, but not limited to, AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD 142), MICA, MICB, LRP1 (also known as CD 91), HMGB1, ABO, RHD, FUT1, PDGFRa, OLIG2, GFAP, or KDM5D gene locus. In some embodiments, the polynucleotide encoding the tolerogenic factor is inserted into the B2M gene locus, CIITA gene locus, TRAC gene locus or TRB gene locus. In some embodiments, the polynucleotide encoding the tolerogenic factor is inserted into any of the genetic loci described in table 4 provided herein. In certain embodiments, the polynucleotide encoding a tolerogenic factor is operably linked to a promoter.

O, gene modification method

In some embodiments, the rare-cutting endonuclease is introduced into a cell comprising a polynucleotide sequence of interest in the form of a nucleic acid encoding the rare-cutting endonuclease. The process of introducing the nucleic acid into the cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid mediated transfection, electroporation and transduction or infection with viral vectors. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises modified DNA, as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises a modified mRNA, as described herein (e.g., synthetic, modified mRNA).

The gene editing systems of the present disclosure (e.g., CRISPR/Cas) can be used to alter the target polynucleotide sequences described herein in any manner available to a person of ordinary skill. Any CRISPR/Cas system capable of altering a polynucleotide sequence of interest in a cell can be used. This CRISPR/Cas system can use a variety of Cas proteins (Haft et al, PLoS Comput biol.2005;1 (6) e 60). Molecular mechanisms of this Cas protein that allow CRISPR/Cas systems to alter target polynucleotide sequences in cells include RNA binding proteins, endonucleases, exonucleases, helicases, and polymerases. In some embodiments, the CRISPR/Cas system is a CRISPR type I system. In some embodiments, the CRISPR/Cas system is a CRISPR type II system. In some embodiments, the CRISPR/Cas system is a CRISPR type V system.

The gene editing (e.g., CRISPR/Cas) systems disclosed herein can be used to alter any polynucleotide sequence of interest in a cell. One of ordinary skill in the art will readily appreciate that a polynucleotide sequence of interest to be altered in any particular cell may correspond to any genomic sequence (where expression of the genomic sequence is associated with a deregulation or aids in entry of a pathogen into the cell). For example, a desired target polynucleotide sequence that is altered in a cell can be a polynucleotide sequence that corresponds to a genomic sequence (including a disease associated with a single polynucleotide polymorphism). In such examples, the CRISPR/Cas systems disclosed herein can be used to correct SNPs associated with disease in a cell by substitution with wild-type dual genes. As another example, the polynucleotide sequence of a target gene responsible for entry or proliferation of a pathogen may be the appropriate target for deletion or insertion to disrupt the function of the target gene, avoiding entry or proliferation of the pathogen within the cell.

In some embodiments, the polynucleotide sequence of interest is a genomic sequence. In some embodiments, the polynucleotide sequence of interest is a human genomic sequence. In some embodiments, the polynucleotide sequence of interest is a mammalian genomic sequence. In some embodiments, the polynucleotide sequence of interest is a vertebrate genomic sequence.

In some embodiments, a CRISPR/Cas system provided herein includes a Cas protein and at least one to two ribonucleic acids capable of guiding the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues (i.e., a polymer of amino acids) joined by peptide bonds, and include modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, fragments, and other equivalents, variants, and analogs of the foregoing.

In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some examples, the substitution and/or modification may avoid or reduce proteolytic degradation in the cell and/or extend the half-life of the polypeptide. In some embodiments, the Cas protein may comprise peptide bond substitutions (e.g., urea, thiourea, urethane, sulfonylurea, etc.). In some embodiments, the Cas protein may comprise naturally occurring amino acids. In some embodiments, the Cas protein may comprise a surrogate amino acid (e.g., D-amino acid, β -amino acid, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications to include moieties (e.g., pegylation, glycosylation, lipidation, acetylation, capping, etc.).

In some embodiments, the Cas protein comprises a core Cas protein, an isoform thereof, or any Cas-like protein having similar function or activity of any Cas protein or isoform thereof. Exemplary Cas core proteins include, but are not limited to, cas1, cas2, cas3, cas4, cas5, cas6, cas7, cas8, and Cas9. In some embodiments, the Cas protein comprises a Cas protein of the e.coli subtype (also known as CASS 2). Exemplary Cas proteins of e.coli subtypes include, but are not limited to Cse1, cse2, cse3, cse4, and Cas5e. In some embodiments, the Cas protein comprises a Cas protein of the Ypest subtype (also known as CASS 3). Exemplary Cas proteins of the Ypest subtype include, but are not limited to, csy1, csy2, csy3, and Csy4. In some embodiments, the Cas protein comprises a Cas protein of the nmei subtype (also known as CASS 4). Exemplary Cas proteins for the nmei subtype include, but are not limited to, csn1 and Csn2. In some embodiments, the Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS 1). Exemplary Cas proteins of the Dvulg subtype include Csd1, csd2 and Cas5d. In some embodiments, the Cas protein comprises a Tneap subtype Cas protein (also known as CASS 7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, cst1, cst2, cas5t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of Hmari subtypes include, but are not limited to Csh1, csh2, and Cas5h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (also known as CASS 5). Exemplary Cas proteins of the Apern subtype include, but are not limited to Csa1, csa2, csa3, csa4, csa5, and Cas5a. In some embodiments, the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS 6). Exemplary Cas proteins of the Mtube subtype include, but are not limited to, csm1, csm2, csm3, csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, cmr1, cmr2, cmr3, cmr4, cmr5, and Cmr6. See, e.g., klompe et al, nature 571,219-225 (2019); strecker et al, science 365,48-53 (2019). In some embodiments, the Cas protein comprises a Cas protein of subtype I. Type I CRISPR/Cas effector proteins are a subtype of type 1 CRISPR/Cas effector proteins. Examples include, but are not limited to: cas3, cas8a, cas5, cas8b, cas8c, cas10d, cse1, cse2, csy1, csy2, csy3 and/or GSU0054. In some embodiments, the Cas protein comprises Cas3, cas8a, cas5, cas8b, cas8c, cas10d, cse1, cse2, csy1, csy2, csy3, and/or GSU0054. In some embodiments, the Cas protein comprises a Cas protein of subtype II. Type II CRISPR/Cas effector proteins are subtypes of type 2 CRISPR/Cas effector proteins. Examples include, but are not limited to: cas9, csn2, and/or Cas4. In some embodiments, the Cas protein comprises Cas9, csn2, and/or Cas4. In some embodiments, the Cas protein comprises a Cas protein of subtype III. Type III CRISPR/Cas effector proteins are subtypes of type 1 CRISPR/Cas effector proteins. Examples include, but are not limited to: cas10, csm2, cmr5, cas10, csx11, and/or Csx10. In some embodiments, the Cas protein comprises Cas10, csm2, cmr5, cas10, csx11, and/or Csx10. In some embodiments, the Cas protein comprises a Cas protein of subtype IV. Type IV CRISPR/Cas effector proteins are subtypes of type 1 CRISPR/Cas effector proteins. Examples include, but are not limited to: csf1. In some embodiments, the Cas protein comprises Csf1. In some embodiments, the Cas protein comprises a Cas protein of subtype V. The type V CRISPR/Cas effector protein is a subtype of type 2 CRISPR/Cas effector protein. For examples of type V CRISPR/Cas systems and their effector proteins (e.g., cas12 family proteins such as Cas12 a), see, e.g., shmakov et al, nat Rev microbiol 2017;15 (3): 169-182: "Diversity and evolution of class 2CRISPR-Cas systems". Examples include, but are not limited to: cas12 family (Cas 12a, cas12b, cas 12C), C2C4, C2C8, C2C5, C2C10, and C2C9; and CasX (Cas 12 e) and CasY (Cas 12 d). See also, for example, koonin et al, curr Opin microbiol 2017;37:67-78: "Diversity, classification and evolution of CRISPR-Cas systems". In some embodiments, the Cas protein comprises a Cas12 protein, such as Cas12a, cas12b, cas12c, cas12d, and/or Cas12e.

In some embodiments, the Cas protein comprises any one of the Cas proteins or functional portions thereof described herein. As used herein, "functional moiety" refers to a portion of a peptide that encompasses the ability to complex with at least one ribonucleic acid (e.g., guide RNA (gRNA)) and cleave a polynucleotide sequence of interest. In some embodiments, the functional moiety comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of: a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional moiety comprises a combination of operably linked Cas12a (also known as Cpf 1) protein functional domains selected from the group consisting of: a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, the functional portion of the Cas9 protein comprises a RuvC domain-like functional portion. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of an HNH nuclease domain. In some embodiments, the functional portion of the Cas12a protein comprises a RuvC domain-like functional portion.

In some embodiments, the exogenous Cas protein may be introduced into the cell in the form of a polypeptide. In certain embodiments, the Cas protein may be conjugated or fused to a cell-penetrating polypeptide or a cell-penetrating peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refer to a polypeptide or peptide, respectively, that facilitates uptake of a molecule into a cell. The cell-penetrating polypeptide may comprise a detectable marker.

In certain embodiments, the Cas protein may be conjugated to or fused to a charged protein (e.g., positively charged, negatively charged, or overall neutral charge). This linkage may be covalent. In some embodiments, the Cas protein may be fused to a superpositioned GFP to significantly increase the ability of the Cas protein to penetrate cells (Cronican et al ACS Chem biol.2010;5 (8): 747-52). In certain embodiments, the Cas protein may be fused to a Protein Transduction Domain (PTD) to assist in its entry into a cell. Exemplary PTDs include Tat, oligoarginine and transmembrane peptides (penearatins). In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a transmembrane peptide (penetratin) domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a GFP that is superpositively charged. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a PTD. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a tat domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to an oligoarginine domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a transmembrane peptide (penetratin) domain. In some embodiments, the Cas12a protein comprises a Cas12a polypeptide fused to a GFP that is positively charged.

In some embodiments, the Cas protein may be introduced into a cell comprising a polynucleotide sequence of interest in the form of a nucleic acid encoding the Cas protein. The process of introducing nucleic acid into a cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid mediated transfection, electroporation and transduction or infection with viral vectors. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises a modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises a modified mRNA (e.g., synthetic, modified mRNA) as described herein.

In some embodiments, the Cas protein is complexed with 1 to 2 ribonucleic acids. In some embodiments, the Cas protein is complexed with 2 ribonucleic acids. In some embodiments, the Cas protein is complexed with 1 ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., synthetic, modified mRNA) as described herein.

The methods disclosed herein contemplate the use of any ribonucleic acid capable of directing Cas protein to and hybridization to a target motif of a target polynucleotide sequence. In some embodiments, the at least one ribonucleic acid comprises a tracrRNA. In some embodiments, the at least one ribonucleic acid comprises CRISPR RNA (crRNA). In some embodiments, the single ribonucleic acid comprises a guide RNA that guides the Cas protein to and hybridizes to a target motif of a target polynucleotide sequence in a cell. In some embodiments, the at least one ribonucleic acid comprises a guide RNA that guides the Cas protein to and hybridizes to a target motif of a target polynucleotide sequence in the cell. In some embodiments, both 1 to 2 ribonucleic acids comprise guide RNAs that guide Cas proteins to and hybridize to target motifs of target polynucleotide sequences in cells. As will be appreciated by those of ordinary skill in the art, the ribonucleic acids provided herein can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR/Cas system and sequence of the target polynucleotide used. 1 to 2 ribonucleic acids may also be selected to minimize hybridization to nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, 1 to 2 ribonucleic acids hybridize to a target motif comprising at least two errors when compared to genomic nucleotide sequences in all other cells. In some embodiments, 1 to 2 ribonucleic acids hybridize to a cell comprising at least one misplaced motif of interest when compared to genomic nucleotide sequences in all other cells. In some embodiments, 1 to 2 ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleotide motif recognized by a Cas protein. In some embodiments, 1 to 2 ribonucleic acids each are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleotide motif recognized by a Cas protein, sandwiching a mutant pair gene located between the target motifs.

In some embodiments, each of the 1 to 2 ribonucleic acids comprises a guide RNA that guides Cas protein to and hybridizes to a target motif of a target polynucleotide sequence in a cell.

In some embodiments, one or both ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to sequences on the same strand of the polynucleotide sequence of interest. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to sequences on opposite strands of a polynucleotide sequence of interest. In some embodiments, one or both ribonucleic acids (e.g., guide RNAs) are not complementary to and/or hybridized to sequences on opposite strands of the polynucleotide sequence of interest. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to overlapping target motifs of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and/or hybridize to a complementing target motif of a target polynucleotide sequence.

In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding at least one to two ribonucleic acids are introduced into the cell by viral transduction (e.g., lentiviral transduction). In some embodiments, the Cas protein is complexed with 1 to 2 ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., synthetic, modified mRNA) as described herein.

Table 2 provides exemplary gRNA sequences for CRISPR/Cas-based targeting of genes described herein. The sequence may be found in WO2016183041, filed 5/9/2016, the disclosure of which, including tables, attachments and sequence listings, is incorporated herein by reference in its entirety.

TABLE 2 exemplary gRNA sequences for targeting genes

Other exemplary gRNA sequences for CRISPR/Cas-based targeting of the genes described herein are provided in U.S. provisional patent application No. 63/190,685 to 2021, 5 month 19 and U.S. provisional patent application No. 63/221,887 to 2021, 7 month 14, the disclosures of which, including tables, appendages, and sequence listings, are incorporated herein by reference in their entirety.

In some embodiments, the cells described herein are made using a transcription activator-like effector nuclease (TALEN) method. "TALE-nuclease" (TALEN) means a fusion protein consisting of a nucleic acid-binding domain typically derived from a transcription activator-like effector (TALE) and a nuclease catalytic domain that cleaves a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, such as, for example, I-TevI, colE7, nucA and Fok-I. In a particular embodiment, the TALE domain can be fused to meganucleases such as, for example, I-CreI and I-OnuI or functional variants thereof. In a more preferred embodiment, the nuclease is a monomeric TALE-nuclease. Monomeric TALE-nucleases are fusions of engineered TAL repeats with the catalytic domain of I-TevI that do not require dimerization for specific recognition and cleavage, such as described in WO 2012138927. A transcription activator-like effector (TALE) is a protein from the bacterial species Xanthomonas (Xanthomonas), comprising multiple repeats, each repeat comprising a diradical (RVD) at positions 12 and 13, specific for each nucleotide base of the sequence targeted by the nucleic acid. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBD) can also be derived from novel modular proteins of different bacterial species recently discovered by applicant. The novel modular proteins have the advantage of exhibiting more sequence variability than TAL repeats. Preferably, the RVDs associated with the identification of the different nucleotides are HD for identifying C, NG for identifying T, NI for identifying a, NN for identifying G or a, NS for identifying A, C, G or T, HG for identifying T, IG for identifying T, NK for identifying G, HA for identifying C, ND for identifying C, HI for identifying C, HN for identifying G, NA for identifying G, SN for identifying G or a and YG for identifying T, TL for identifying a, VT for identifying a or G and SW for identifying a. In another embodiment, amino acids 12 and 13 may be mutated to other amino acid residues to modulate their specificity for nucleotides A, T, C and G and in particular, to increase this specificity. TALEN kits are commercially available.

In some embodiments, the cells are manipulated using Zinc Finger Nucleases (ZFNs). Since the protein structure is stabilized by coordination of zinc ions, a "zinc finger binding protein" is a protein or polypeptide that binds DNA, RNA and/or protein, preferably in a sequence-specific manner. The term zinc finger binding protein is commonly abbreviated as zinc finger protein or ZFP. Individual DNA binding domains are typically referred to as "fingers". ZFP has at least one finger, typically 2 fingers, 3 fingers, or 6 fingers. Each finger binds between 2 and 4 base pairs of DNA, typically 3 or 4 base pairs of DNA. ZFP binds to a nucleic acid sequence called a target site or segment. Each finger typically comprises about 30 amino acids, zinc chelate, DNA binding subdomain. Studies have demonstrated that such single zinc fingers consist of an alpha helix comprising two invariant histidine residues coordinated to zinc, together with two cysteine residues of a single beta turn (see, e.g., berg & Shi, science271:1081-1085 (1996)).

In some embodiments, the cells described herein are made using homing endonucleases. Such homing endonucleases are known in the art (Stoddard 2005). Homing endonucleases recognize DNA target sequences and create single or double strand breaks. Homing endonucleases are highly specific, recognizing DNA target sites, ranging from 12 to 45 base pairs (bp) in length, typically ranging from 14 to 40bp in length. The homing endonuclease may for example correspond to a LAGLIDADG endonuclease, to an HNH endonuclease or to a GIY-YIG endonuclease. In some embodiments, the homing endonuclease can be an I-CreI variant.

In some embodiments, the cells described herein are made using meganucleases. Meganucleases, as their name implies, are sequence-specific endonucleases (chemalier, b.s.and b.l.stoddard, nucleic Acids res.,2001,29,3757-3774) that recognize large sequences. They can cleave unique sites in living cells, thereby enhancing gene targeting in the vicinity of the cleavage site 1000-fold or more (Puchta et al, nucleic Acids Res.,1993,21,5034-5040; rouet al, mol.cell.biol.,1994,14,8096-8106; choulika et al, mol.cell.biol.,1995,15,1968-1973; puchta et al, proc.Natl.Acad.Sci. USA,1996,93,5055-5060; sargent et al, mol.cell.biol.,1997,17,267-77; donoho et al, mol.cell.biol.,1998,18,4070-4078; elliott et al, mol.cell.biol.,1998,18,93-101; cohen-Tannoudji et al, mol.cell.biol.,1998,18,1444-1448).

In some embodiments, the cells provided herein are made using RNA silencing or RNA interference (RNAi, also known as siRNA) to attenuate (e.g., reduce, eliminate, or inhibit) expression of a polypeptide, such as a tolerogenic factor. Useful RNAi methods include those utilizing synthetic RNAi molecules, short interfering RNAs (siRNAs), PIWI-interacting NRAs (piRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), and transient attenuation methods recognized by those of ordinary skill in the art to which the invention pertains. RNAi agents, including sequence-specific shRNA, siRNA, miRNA and the like, are commercially available. For example, CIITA can be attenuated in pluripotent stem cells by introducing CIITA siRNA or transducing CIITA shRNA-expressing viruses into the cells. In some embodiments, RNA interference is employed to reduce or inhibit expression of at least one selected from the group consisting of: CIITA, B2M and NLRC5.

1. Gene editing system

In some embodiments, methods of genetically modifying cells to knock out, attenuate, or modify one or more genes comprise the use of site-directed nucleases, including, for example, zinc Finger Nucleases (ZFNs), transcription-like activator-effector nucleases (TALENs), meganucleases, translocases, and Clustered and Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems, as well as nickase (nickase) systems, base editing systems, primary editing systems, and gene writing systems, as known in the art of the invention.

a)ZFN

ZFNs are fusion proteins comprising a series of site-specific DNA binding domains, tailored from zinc-finger-containing transcription factors attached to the endonuclease domains of bacterial fokl restriction enzymes. ZFNs can have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) DNA binding domains or zinc finger domains. See, for example, carroll et al Genetics Society of America (2011) 188:773-782; kim et al Proc.Natl.Acad.Sci.USA (1996) 93:1156-1160. Each zinc finger domain is a small protein structural motif stabilized by one or more zinc ions and typically recognizes 3 to 4-bp DNA sequences. Thus, tandem domains can potentially bind to uniquely extended nucleotide sequences in the cell genome.

Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize about 6, 9, 12, 15, or 18-bp sequences. Various selection and module assembly techniques are available to generate zinc fingers (and combinations thereof) that recognize specific sequences, including phage display, yeast-hybridization systems, bacterial-hybridization and-hybridization systems, and mammalian cells. The zinc fingers can be engineered to bind to a predetermined nucleic acid sequence. Criteria for engineering zinc fingers to bind to predetermined nucleic acid sequences are known in the art. See, e.g., sera et al, biochemistry (2002) 41:7074-7081; liu et al, bioenformatics (2008) 24:1850-1857.

ZFNs comprising fokl nuclease domains or other dimeric nuclease domains act as dimers. Thus, a pair of ZFNs is required to target non-palindromic DNA sites. Two individual ZFNs must bind opposite strands of DNA, appropriately spaced from their nucleases. See Bitinaite et al, proc.Natl. Acad.Sci.USA (1998) 95:10570-10575. To cleave a specific site in the genome, a pair of ZFNs was designed to recognize two sequences flanking the site, one on the forward strand and the other on the reverse strand. Upon ZFN binding on either side of the site, the nuclease domain dimerizes and cleaves DNA at the site, producing a DSB with a 5' overhang. HDR can then be used to introduce specific mutations, with the help of repair templates, containing the desired mutations, flanked by homology arms. Repair templates are typically exogenous double-stranded DNA vectors that are introduced into cells. See se Miller et al, nat. Biotechnol (2011) 29:143-148; hockmeyer et al, nat. Biotechnol (2011) 29:731-734.

b)TALEN

TALENs are another example of artificial nucleases that can be used to edit a gene of interest. TALENs are DNA binding domains derived from so-called TALE repeats, which typically comprise a tandem array of 10 to 30 repeats that bind and recognize an extended DNA sequence. Each repeat is 33 to 35 amino acids in length, having two adjacent amino acids (referred to as repeat variable diradicals, or RVDs), conferring specificity to one of the four DNA base pairs. Thus, there is a one-to-one correspondence between repeats and base pairs in the target DNA sequence.

TALENs are created artificially by fusing one or more TALE DNA binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) with a nuclease domain (e.g., a fokl endonuclease domain). See Zhang, nature biotech (2011) 29:149-153. Several mutations have been made to fokl for TALENs; these, for example, improve cleavage specificity or activity. See cerak et al, nucleic acids res (2011) 39: e82; miller et al Nature Biotech. (2011) 29:143-148; hockmeyer et al Nature biotech (2011) 29:731-734; wood et al, science (2011) 333: 307. Doyon et al, nature Methods (2010) 8:74-79; szczepek et al, nature Biotech (2007) 25:786-793; guo et al, j.mol.biol. (2010) 200:96. the fokl domain acts as a dimer, requiring two constructs with unique DNA binding domains for sites in the genome of interest with the proper orientation and spacing. The number of amino acid residues between the TALE DNA binding domain and the fokl nuclease domain, and the number of bases between two individual TALEN binding sites, appear to be important parameters to achieve high amounts of activity. Miller et al Nature Biotech. (2011) 29:143-148.

By combining the engineered TALE repeat sequence with a nuclease domain, a site-specific nuclease for any desired DNA sequence can be specifically generated. Like ZFNs, TALENs can be introduced into cells to produce DSBs at desired target sites in the genome, and thus can be used to knock out genes or knock-in mutations in a similar, HDR-mediated pathway. See bosh, nature biotech (2011) 29:135-136; boch et al, science (2009) 326:1509-1512; moscou et al, science (2009) 326:3501.

c) Meganucleases

Meganucleases are enzymes belonging to the family of endonucleases, characterized by their ability to recognize and cleave large DNA sequences (14 to 40 base pairs). Meganucleases are divided into families according to their structural motifs that affect nuclease activity and/or DNA recognition. The most widespread and well known meganucleases are proteins in the LAGLIDADG family, their names being attributed to the retained amino acid sequence. See Chevalier et al, nucleic Acids Res. (2001) 29 (18): 3757-3774. On the other hand, GIY-YIG family members have GIY-YIG modules, which are 70 to 100 residues long and include four or five conserved sequence motifs with four invariant residues, two of which are essential for activity. See Van rouy et al, nature struct. Biol. (2002) 9:806-811.His-Cys family meganucleases are characterized by a highly conserved histidine and cysteine series over a region encompassing hundreds of amino acid residues. See Chevalier et al, nucleic Acids Res. (2001) 29 (18): 3757-3774. Members of the NHN family are defined by motifs that comprise two pairs of retained histidines surrounded by asparagine residues. See Chevalier et al, nucleic Acids Res. (2001) 29 (18): 3757-3774.

Because of the low chance of identifying natural meganucleases for a particular target DNA sequence due to high specificity requirements, various methods including mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Strategies for engineering meganucleases with altered DNA binding specificity, e.g., binding to a predetermined nucleic acid sequence, are known in the art to which the invention pertains. See, e.g., chevalier et al, mol.cell. (2002) 10:895-905; epinat et al, nucleic Acids Res (2003) 31:2952-2962; silva et al, J mol. Biol. (2006) 361:744-754; seligman et al, nucleic Acids Res (2002) 30:3870-3879; sussman et al, J mol biol (2004) 342:31-41; doyon et al, J Am Chem Soc (2006) 128:2477-2484; chen et al, protein Eng Des Sel (2009) 22:249-256; arnoul d et al, J Mol biol. (2006) 355:443-458; smith et al, nucleic Acids Res. (2006) 363 (2): 283-294.

Like ZFNs and TALENs, meganucleases can produce DSBs in genomic DNA, and if improperly repaired, can create frame shift mutations, e.g., through NHEJ, resulting in reduced expression of the target gene in the cell. Alternatively, foreign DNA may be introduced into the cell along with the meganuclease. Depending on the sequence of the foreign DNA and the chromosomal sequence, this process can be used to modify the gene of interest. See Silva et al, current Gene Therapy (2011) 11:11-27.

d) Translocase enzyme

Translocases are enzymes that bind to the ends of a translocation molecule and catalyze its movement to another part of the genome by a cleavage and adhesion mechanism or a replication translocation mechanism. By linking the translocases to other systems, such as the CRISPER/Cas system, new gene editing tools can be developed to achieve site-specific insertion or manipulation of genomic DNA. There are two known methods of DNA integration using a transposon that uses a catalytically inactive Cas effector protein and a Tn 7-like transposon. The transposase dependent DNA integration does not cause genomic DSB, which can guarantee safer and more specific DNA integration.

e) CRISPR/Cas system

CRISPR systems were originally found in prokaryotic organisms (e.g., bacteria and archaea) as systems involving phages and plasmids that are protected from invasion, which provide a form of acquired immunity. It has now been adapted and used as a popular gene editing tool in research and clinical applications.

CRISPR/Cas systems generally comprise at least two components: one or more guide RNAs (grnas) and a Cas protein. Cas proteins are nucleases that introduce DSBs into the site of interest. CRISPR-Cas systems fall into two broad categories: class 1 systems use complexes of multiple Cas proteins to degrade nucleic acids; class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into class I, III and class IV; class 2 is divided into classes II, V and VI. Different Cas proteins suitable for use in gene editing applications include, but are not limited to, cas3, cas4, cas5, cas8a, cas8b, cas8C, cas9, cas10, cas12a (Cpf 1), cas12b (C2C 1), cas12C (C2C 3), cas12d (CasY), cas12e (CasX), cas12f (C2C 10), cas12g, cas12h, cas12i, cas12k (C2C 5), cas13a (C2), cas13b, cas13C, cas13d, C2C4, C2C8, C2C9, cmr5, cse1, cse2, csf1, csm2, csn2, csx10, csx11, csy1, csy2, csy3, and Mad7. The most widely used Cas9 is a type II Cas protein and is described herein as an illustration. These Cas proteins may be derived from different source species. For example, cas9 may be derived from staphylococcus pyogenes or staphylococcus aureus.

In the original microbial genome, a type II CRISPR system incorporates sequences from invasive DNA within the host genome between CRISPR repeats encoded as an array. Transcripts from the CRISPR repeat array are processed into CRISPR RNA (crRNA), each possessing a variable sequence transcribed from the invaded DNA, termed a "protospacer" sequence, and a portion of the CRISPR repeat. Each crRNA hybridizes to a second transactivation CRISPR RNA (tracrRNA), and both RNAs form a complex with the Cas9 nuclease. The portion of the crRNA encoded by the protospacer directs the Cas9 complex to cleave the complementary DNA sequence of interest, provided that they are adjacent to a short sequence called a "protospacer adjacent motif" (PAM).

Since discovery, CRISPR systems have been adapted for inducing sequence-specific DSBs and target genome editing in a wide range of cells and organisms ranging from bacteria to eukaryotic cells (including human cells). For use in gene editing applications, artificially designed, synthetic grnas have replaced the original crrnas: tracrRNA complex. For example, the gRNA can be a single guide RNA (sgRNA), consisting of crRNA, tetracyclic and tracrRNA. crrnas typically contain complementary regions (also referred to as spacers, typically about 20 nucleotides in length) designed for the user to recognize the target DNA of interest. the tracrRNA sequence comprises a scaffold region for Cas nuclease binding. The crRNA sequence and the tracrRNA sequence are joined by four loops and each have a short repeat sequence for hybridization to each other, thereby producing a chimeric sgRNA. The genomic target of the Cas nuclease can be altered by simply altering the spacer or complementary region sequences present in the gRNA. The complementary region will direct the Cas nuclease to the target DNA site by standard RNA-DNA complementary base pairing rules.

In order for Cas nuclease to function, there must be PAM immediately downstream of the target sequence in the genomic DNA. PAM recognition by Cas proteins is believed to destabilize adjacent genomic sequences, allowing interrogation by the sequence of the gRNA and resulting in gRNA-DNA pairing when a matching sequence is present. The specific sequence of PAM varies with the species of Cas gene. For example, the most commonly used Cas9 nucleases derived from staphylococcus suppurative recognize PAM sequences of 5'-NGG-3', or less efficient 5'-NAG-3', where "N" can be any nucleotide. Other Cas nuclease variants with alternative PAMs have also been characterized and successfully used for genome editing, summarized in table 3 below.

TABLE 3 exemplary Cas nuclease variants and PAM sequences thereof

CRISPR nuclease	Source organism	PAM sequence (5 '. Fwdarw.3')
			SpCas9	Staphylococcus suppurative	NGG or NAG
SaCas9	Staphylococcus aureus	NGRRT or NGRRN
			NmeCas9	Meningococci	NNNNGATT
CjCas9	Campylobacter jejuni	NNNNRYAC
			StCas9	Streptococcus thermophilus	NNAGAAW
TdCas9	Dense tooth scale screw	NAAAAC
			LbCas12a(Cpf1)	Bacteria of the family Maotaceae	TTTV
AsCas12a(Cpf1)	Amino acid ballGenus of bacteria	TTTV
			AacCas12b	Acidophilic alicyclic acid bacillus	TTN
BhCas12b v4	Bacillus stearothermophilus	ATTN, TTTN or GTTN

R=a or G; y=c or T; w=a or T; v=a or C or G; n=any base

In some embodiments, the Cas nuclease may comprise one or more mutations to alter its activity, specificity, recognition, and/or other characteristics. For example, a Cas nuclease may have one or more mutations that alter its fidelity to mitigate off-target effects (e.g., eSpCas9, spCas9-HF1, hypascas 9, heFSpCas9, and evoSpCas9 high fidelity variants of SpCas 9). As another example, a Cas nuclease may have one or more mutations that alter its PAM specificity.

In some embodiments, the cells provided herein are genetically modified to reduce expression of one or more immune factors (including a polypeptide of interest) to create immune-privileged or low-immunity cells. In certain embodiments, the cells disclosed herein (e.g., stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary T cells, and CAR-T cells) comprise one or more genetic modifications to reduce expression of one or more polynucleotides of interest. Non-limiting examples of such polynucleotides and polypeptides of interest include CIITA, B2M, NLRC, CTLA4, PD1, HLA-A, HLA-BM, HLA-C, RFX-ANK, NFY-A, RFX5, RFX-AP, NFY-B, NFY-C, IRF1 and/or TAP1.

In some embodiments, the genetic modification occurs using a CRISPR/Cas system. By modulating (e.g., reducing or deleting) the expression of one or more polynucleotides of interest, the cell exhibits reduced immune activation when implanted into a recipient subject. In some embodiments, the cells are considered to be hypoimmunity, e.g., in a recipient subject or patient after administration.

f) Nickase (nicase)

The nuclease domain of Cas, particularly Cas9, can be independently mutated to produce an enzyme called DNA "nickase". Nickases are able to introduce single strand cleavage with the same specificity as conventional CRISPR/Cas nuclease systems (including, for example, CRISPR/Cas 9). Nicking enzymes can be used to create double strand breaks that can be used in gene editing systems (Mali et al, nat Biotech,31 (9): 833-838 (2013); mali et al, nature Methods,10:957-963 (2013); mali et al, science,339 (6121): 823-826 (2013)). In some examples, when two Cas nickases are used, long overhangs are created at each of the cleavage ends, rather than at each of the blunt ends, which allows for additional control over precise gene integration and insertion (Mali et al, nat Biotech,31 (9): 833-838 (2013); mali et al, nature Methods,10:957-963 (2013); mali et al, science,339 (6121): 823-826 (2013)). Since both nicking Cas enzymes must efficiently cleave their target DNA, paired nicking enzymes have lower off-target effects than double-stranded nicking Cas-based systems (Ran et al, cell,155 (2): 479-480 (2013); mali et al, nat Biotech,31 (9): 833-838 (2013); mali et al, nature Methods,10:957-963 (2013); mali et al, science,339 (6121): 823-826 (2013)).

Method for recombinant expression of tolerogenic factors and/or chimeric antigen receptors

For all of these techniques, well-known recombinant techniques are used to generate the recombinant nucleic acids outlined herein. In certain embodiments, the recombinant nucleic acid encodes one or more regulatory nucleotide sequences operably linked to the tolerogenic factors or chimeric antigen receptor in the expression construct. Regulatory nucleotide sequences are generally suitable for the host cell and recipient subject to be treated. For a variety of host cells, many types of suitable expression vectors and suitable regulatory sequences are known in the art to which the invention pertains. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art are also contemplated. The promoter may be a naturally occurring promoter, or a hybrid promoter combining elements of more than one promoter. The expression construct may be present on the cell on an episome, such as a plasmid, or the expression construct may be inserted into a chromosome. In a particular embodiment, the expression vector includes a selectable marker gene to allow selection of transformed host cells. Certain embodiments include expression vectors comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequences as used herein include promoters, enhancers and other expression control elements. In certain embodiments, the expression vector is designed for selection of the host cell to be transformed, the particular variant polypeptide to be expressed, the copy number of the vector, the ability to control the copy number, and/or expression of any other protein encoded by the vector, such as an antibiotic marker.

Examples of suitable mammalian promoters include, for example, promoters from the following genes: elongation factor 1α (EF 1 α) promoter, hamster ubiquitin/S27 a promoter (WO 97/15664), monkey vacuolated virus 40 (SV 40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, terminal long repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), moloney murine leukemia virus terminal long repeat region, and human Cytomegalovirus (CMV) early promoter. Examples of other heterologous mammalian promoters are actin, immunoglobulin or heat shock promoters. In additional embodiments, the promoter for the mammalian host cell may be obtained from the genome of a virus, such as polyomavirus, avipoxvirus (UK 2,211,504 disclosed in 7.5.1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and monkey virus 40 (SV 40). In a further embodiment, a heterologous mammalian promoter is used. Examples include actin promoters, immunoglobulin promoters and heat shock promoters. The early and late promoters of SV40 are conveniently obtained as SV40 restriction fragments that also contain the SV40 viral origin of replication (Fiers et al Nature 273:113-120 (1978)). The earliest promoter of human cytomegalovirus is conveniently obtained as a HindIII restriction enzyme fragment (Greenaway et al, gene 18:355-360 (1982).

In some embodiments, the expression vector is a bicistronic or polycistronic expression vector. A bicistronic or polycistronic expression vector can comprise (1) a multiple promoter fused to each of the open reading frames; (2) insertion of splicing signals between genes; (3) expressing a gene fusion driven by a single promoter; and (4) insertion of a proteolytic cleavage site (self-cleaving peptide) between genes or insertion of an Internal Ribosome Entry Site (IRES) between genes.

The process of introducing the polynucleotides described herein into a cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid mediated transfection, electroporation, fusion (fusogen) and transduction or infection with viral vectors. In some embodiments, the polynucleotide is introduced into the cell by viral transduction (e.g., lentiviral transduction) or delivery in a viral vector (e.g., fusion-mediated delivery).

Provided herein are cells that do not trigger or activate an immune response upon administration to a recipient subject. As described above, in some embodiments, the cells are modified to increase expression of genes and tolerogenic (e.g., immune) factors that affect immune recognition and tolerance in the recipient.

In certain embodiments, any of the cells disclosed herein that possess genomic modifications that modulate the expression of one or more proteins of interest listed herein (e.g., stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary T cells CAR-T cells, and CAR-NK cells) are also modified to express one or more tolerogenic factors. Exemplary tolerogenic factors include, without limitation, one or more of CD47, DUX4, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, fasL, CCL21, CCL22, mfge8 and Serphinb 9. In some embodiments, the tolerogenic agent is selected from the group consisting of: DUX4, CD47, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, fasL, CCL21, CCL22, mfge8 and Serphinb 9.

Useful genomic, polynucleotide and polypeptide information about human CD27 (also known as CD27L receptor, tumor necrosis factor receptor superfamily member 7 (TNFSF 7), T cell activating antigens S152, tp55, and T14) is provided, for example, in GeneCard Identifier GC P008144, HGNC No.11922, NCBI Gene ID 939, uniprot No. P26842, and NCBI RefSeq nos. nm_001242.4 and np_001233.1.

Useful genomic, polynucleotide and polypeptide information about human CD46 is provided, for example, in GeneCard Identifier GC P207752, HGNC No.6953, NCBI Gene ID 4179, uniprot No. P15529 and NCBI RefSeq nos. nm_002389.4, nm_153826.3, nm_172350.2, nm_172351.2, nm_172352.2np_75860.1, nm_172353.2, nm_172359.2, nm_172361.2, np_002380.3, np_722548.1, np_758860.1, np_758861.1, np_758862.1, np_758863.1, np_758869.1 and np_758871.1.

Useful genomic, polynucleotide and polypeptide information about human CD55 (also known as complement decay acceleration factor) is provided, for example, in GeneCard Identifier GC P207321, HGNC No.2665, NCBI Gene ID 1604, uniprot No. P08174 and NCBI RefSeq Nos. NM_000574.4, NM_001114752.2, NM_001300903.1, NM_001300904.1, NP_000565.1, NP_001108224.1, NP_001287832.1 and NP_001287833.1.

Useful genomic, polynucleotide and polypeptide information about human CD59 is provided, for example, in GeneCard Identifier GC M033704, HGNC No.1689, NCBI GENE ID 966, uniprot No. p13987 and NCBI RefSeq nos. np_000602.1, nm_000611.5, np_001120695.1, nm_001127223.1, np_001120697.1, nm_001127225.1, np_001120698.1, nm_001127226.1, np_001120699.1, nm_001127227.1, np_976074.1, nm_203329.2, np_976075.1, nm_203330.2, np_976076.1 and nm_203331.2.

Useful genomic, polynucleotide and polypeptide information about human CD200 is provided, for example, in GeneCard Identifier GC P112332, HGNC No.7203, NCBI Gene ID 4345, uniprot No. P41217 and NCBI RefSeq Nos. NP_001004196.2, NM_001004196.3, NP_001305757.1, NM_001318828.1, NP_005935.4, NM_005944.6, XP_005247539.1 and XM_005247482.2.

Useful genomic, polynucleotide, and polypeptide information about human HLA-C is provided, for example, in GeneCard Identifier GC M031272, HGNC No.4933, NCBI Gene ID 3107, uniprot No. P10321, and NCBI RefSeq Nos. NP-002108.4 and NM-002117.5.

Useful genomic, polynucleotide, and polypeptide information about human HLA-E is provided, for example, in GeneCard Identifier GC P047281, HGNC No.4962, NCBI GENE ID 3133, uniprot No. P13747, and NCBI RefSeq Nos. NP-005507.3 and NM-005516.5.

Useful genomic, polynucleotide, and polypeptide information about human HLA-G is provided, for example, in GeneCard Identifier GC P047256, HGNC No.4964, NCBI Gene ID 3135, uniprot No. P17693, and NCBI RefSeq Nos. NP-002118.1 and NM-002127.5.

Useful genomic, polynucleotide and polypeptide information about human PD-L1 or CD274 is provided, for example, in GeneCard Identifier GC09P005450, HGNC No.17635, NCBI Gene ID 29126, uniprot No. q9NZQ7 and NCBI RefSe q Nos. NP-001254635.1, NM-001267706.1, NP-054862.1 and NM-014143.3.

Useful genomic, polynucleotide and polypeptide information about human IDO1 is provided, for example, in GeneCard Identifier GC P039891, HGNC No.6059, NCBI Gene ID 3620, uniprot No. P14202 and NCBI RefSeq nos. np_002155.1 and nm_002164.5.

Useful genomic, polynucleotide and polypeptide information about human IL-10 is provided, for example, in GeneCard Identifier GC M206767, HGNC No.5962, NCBIGene ID 3586, uniprot No. P22301, and NCBI RefSeq Nos. NP-000563.1 and NM-000572.2.

Useful genomic, polynucleotide and polypeptide information about human Fas ligand (also known as FasL, FASLG, CD, TNFSF6, etc.) is provided, for example, in GeneCard Identifier GC P172628, HGNC No.11936, NCBI Gene ID 356, uniprot No. P48023 and NCBI RefSeq Nos. NP-000630.1, NM-000639.2, NP-001289675.1 and NM-001302746.1.

Useful genomic, polynucleotide, and polypeptide information about human CCL21 is provided, for example, in GeneCard Identifier GC M034709, HGNC No.10620, NCB I Gene ID 6366, uniprot No. o00585, and NCBI RefSeq nos. np_002980.1 and nm_002989.3.

Useful genomic, polynucleotide, and polypeptide information about human CCL22 is provided, for example, in GeneCard Identifier GC P057359, HGNC No.10621, NCBI Gene ID 6367, uniprot No. o00626, and NCBI RefSeq nos. np_002981.2, nm_002990.4, xp_016879020.1, and xm_017023531.1.

Useful genomic, polynucleotide and polypeptide information about human Mfge8 is provided, for example, in GeneCard Identifier GC M088898, HGNC No.7036, ncbig ID 4240, uniprot No. q08431 and NCBI RefSeq nos. np_001108086.1, nm_001114614.2, np_001297248.1, nm_001310319.1, np_001297249.1, nm_001310320.1, np_001297250.1, nm_001310321.1, np_005919.2 and nm_005928.3.

Useful genomic, polynucleotide and polypeptide information about human serpin b9 is provided, for example, in GeneCard Identifier GC M002887, HGNC No.8955, NCBI Gene ID 5272, uniprot No. p50453 and NCBI RefSeq nos. np_004146.1, nm_004155.5, xp_005249241.1 and xm_005249184.4.

Methods for modulating the expression of genes and factors (proteins) include genome editing techniques, and RNA or protein expression techniques, among others. For all of these techniques, well-known recombinant techniques are used to generate the recombinant nucleic acids outlined herein.

In some embodiments, expression of a gene of interest (e.g., DUX4, CD47, or another tolerogenic factor) is increased by expression of a fusion protein or protein complex comprising (1) a site-specific binding domain specific for endogenous DUX4, CD47, or other genes and (2) a transcriptional activator.

In some embodiments, the methods are accomplished by genetic modification methods, including homology directed repair/recombination.

In some embodiments, the regulatory factor comprises a site-specific DNA-binding nucleic acid molecule, such as a guide RNA (gRNA). In some embodiments, the method is accomplished by site-specific DNA binding to a targeting protein, such as a Zinc Finger Protein (ZFP) or a fusion protein comprising ZFP, also known as a Zinc Finger Nuclease (ZFN).

In some embodiments, the regulatory factor comprises a site-specific binding domain, such as using a DNA binding protein or DNA binding nucleic acid, that specifically binds to or hybridizes to a gene of interest. In some embodiments, the provided polynucleotides or polypeptides are coupled or complexed with a site-specific nuclease, such as a modified nuclease. For example, in some embodiments, administration is effected using a fusion comprising: modified nuclease DNA-targeting proteins, such as meganucleases or RNA-guided nucleases, such as clustered and regularly interspaced short palindromic nucleic acid (CRISPR) -Cas systems, such as the CRISPR-Cas9 system. In some embodiments, the nuclease is modified to lack nuclease activity. In some embodiments, the modified nuclease is a catalytic death dCas9.

In some embodiments, the site-specific binding domain may be derived from a nuclease. For example, recognition sequences for homing endonucleases and meganucleases, such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. See also U.S. patent No. 5,420,032; U.S. patent No. 6,833,252; belfort et al, (1997) Nucleic Acids Res.25:3379-3388; dujon et al, (1989) Gene 82:115-118; perler et al, (1994) Nucleic Acids Res.22,1125-1127; jasin (1996) Trends Genet.12:224-228; gimble et al, (1996) J.mol. Biol.263:163-180; argast et al, (1998) J.mol. Biol.280:345-353 and new england biology laboratory catalogue. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, e.g., chevalier et al, (2002) molecular cell 10:895-905; epinat et al, (2003) Nucleic Acids Res.31:2952-2962; ashworth et al, (2006) Nature 441:656-659; paques et al, (2007) Current Gene Therapy 7:49-66; U.S. patent publication No. 2007/017128.

The zinc finger, TALE and CRISPR system binding domains may be "engineered" to bind to a predetermined nucleotide sequence, for example by engineering (altering one or more amino acids) a recognition helix region of a naturally occurring zinc finger or TALE protein. The engineered DNA binding protein (zinc finger or TALE) is a non-naturally occurring protein. Reasonable criteria for design include the application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and combined data. See, for example, U.S. patent No. 6,140,081;6,453,242; 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and US publication 20110301073.

In some embodiments, the site-specific binding domain comprises one or more Zinc Finger Proteins (ZFPs) or domains thereof that bind to DNA in a sequence-specific manner. ZFP or a domain thereof is a protein or domain in a larger protein that binds DNA in a sequence-specific manner by one or more zinc fingers, a region of amino acid sequence within the binding domain (the structure of the binding domain is stabilized by coordination of zinc ions).

In ZFP, an artificial ZFP domain, typically 9 to 18 nucleotides long, with a targeting specific DNA sequence is produced by assembly of individual fingers. ZFP includes those in which a single finger domain is about 30 amino acids in length and comprises an alpha helix containing two invariant histidine residues (coordinated by zinc to two cysteines of a single beta turn) and has two, three, four, five or six fingers. In general, the sequence specificity of ZFP can be altered by creating amino acid substitutions at the four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Thus, in some embodiments, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., engineered to bind to a selected target site. See, e.g., beerli et al, (2002) Nature biotechnol.20:135-141; pabo et al, (2001) Ann.Rev.biochem.70:313-340; isalan et al, (2001) Nature biotechnol.19:656-660; segal et al, (2001) curr. Opin. Biotechnol.12:632-637; choo et al, (2000) curr.Opin. Struct.biol.10:411-416; U.S. Pat. nos. 6,453,242;6,534,261;6,599,692;6,503,717;6,689,558;7,030,215;6,794,136;7,067,317;7,262,054;7,070,934;7,361,635;7,253,273; U.S. patent publication 2005/0064474;2007/0218528;2005/0267061, incorporated herein by reference in its entirety.

Many gene-specific engineered zinc fingers are commercially available. For example, sangamo Biosciences (Richmond, CA, USA) in concert with Sigma-Aldrich (St.Louis, MO, USA) developed a zinc finger building platform (CompoZr) that allows researchers to bypass zinc finger building and verification altogether and provide specific targeted zinc fingers for thousands of proteins (Gaj et al Trends in Biotechnology,2013,31 (7), 397-405). In some embodiments, commercially available zinc fingers or custom designs are used.

In some embodiments, the site-specific binding domain comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA binding domain, such as in a transcription activator-like protein effector (TALE) protein, see, e.g., U.S. patent publication No. 20110301073, which is incorporated herein by reference in its entirety.

In some embodiments, the site-specific binding domain is derived from a CRISPR/Cas system. In general, "CRISPR systems" are collectively referred to as transcripts and other elements involved in expressing or directing the activity of a CRISPR-associated ("Cas") gene, including sequences encoding Cas genes, tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or active moiety tracrRNA), tracr-mate sequences (encompassing "guide repeats" and direct repeats of the tracrRNA-treated moiety in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacer" or "targeting sequences" in the context of endogenous CRISPR systems), and/or other sequences and transcripts from CRISPR loci.

In general, the targeting sequence comprises a targeting domain comprising a polynucleotide sequence (having sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding to a CRISPR complex of the target sequence). In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more. In some examples, the targeting domain of the gRNA is complementary, e.g., at least 80, 85, 90, 95, 98, or 99% complementary, e.g., fully complementary, to a target sequence of a target nucleic acid.

In some embodiments, the target site is upstream of the transcription initiation site of the target gene. In some embodiments, the target site is adjacent to a gene transcription initiation site. In some embodiments, the RNA polymerase pause site of the transcription initiation site of the target site gene is downstream adjacent.

In some embodiments, the targeting domain is configured to target a promoter region of a target gene to facilitate transcription initiation, binding of one or more transcription enhancers or activators, and/or RNA polymerase. One or more grnas may be used to target the promoter region of a gene. In some embodiments, one or more regions of a gene may be targeted. In certain aspects, the target site is located within 600 base pairs of either side of the gene Transcription Start Site (TSS).

The design or identification of the gRNA sequences (which are or comprise the sequences of the targeted genes), including the sequences of exons and regulatory regions, including promoters and activators, is within the level of ordinary skill in the art to which the invention pertains. A whole genome gRNA database for CRISPR genome editing is publicly available, which contains an exemplary single guide RNA (sgRNA) target sequence in a constitutive exon of a gene in the human genome or mouse genome (see, e.g., geneescript.com/gRNA-database.html; see also Sanjana et al (2014) Nat. Methods,11:783-4; www.e-crisp.org/E-CRISP/; crispr.mit.edu /). In some embodiments, the gRNA sequence is or comprises a sequence that has minimal off-target binding to a non-target gene.

In some embodiments, the regulatory factor further comprises a functional domain, e.g., a transcriptional activator.

In some embodiments, the transcriptional activator is or comprises one or more regulatory elements, e.g., transcriptional control elements of one or more genes of interest, whereby the site-specific domains provided above are recognized to drive expression of such genes. In some embodiments, the transcriptional activator drives expression of a gene of interest. In some examples, the transcriptional activator may be or comprise all or part of a heterologous transactivation domain. For example, in some embodiments, the transcriptional activator is selected from the group consisting of a herpes simplex-derived transactivation domain, a Dnmt3a methyltransferase domain, p65, VP16, and VP64.

In some embodiments, the regulatory factor is a zinc finger transcription factor (ZF-TF). In some embodiments, the regulatory factor is VP64-p65-Rta (VPR).

In certain embodiments, the regulatory factor further comprises a transcriptional regulatory domain. Common domains include, for example, transcription factor domains (activators, repressors, co-activators, co-repressors), silencers, oncogenes (e.g., myc, jun, fos, myb, max, mad, rel, ets, bcl, myb, mos family members, etc.); DNA repair enzyme and related factors and modifications thereof; DNA rearranging enzyme and related factors and modifications thereof; chromatin-related proteins and modifications thereof (e.g., kinases, acetylases, and deacetylases); and DNA modifying enzymes (e.g., methyltransferases such as members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT L, etc., topoisomerase, helicase, ligase, kinase, phosphatase, polymerase, endonuclease) and related factors and modifications thereof see, e.g., U.S. publication No. 2013/0253040, which is incorporated herein by reference in its entirety.

Suitable domains for achieving activation include the HSV VP 16 activation domain (see, e.g., hagmann et al, J. Virol.71,5952-5962 (1) 97)) nuclear hormone receptor (see, e.g., tochia et al, curr. Opin. Cell. Biol.10:373-383 (1998)); the p65 subunit of the nuclear factor kappa B (Bitko & Bank, J. Virol.72:5610-5618 (1998) and Doyle & Hunt, neuroreport 8:2937-2942 (1997)); liu et al, cancer Gene Ther.5:3-28 (1998)) or artificial chimeric functional domains such as VP64 (Beerli et al, (1998) Proc. Natl. Acad. Sci. USA 95: 14623-33) and degradation determinants (Molinari et al, (1999) EMBO J.18, 6439-6447). Additional exemplary activation domains include Oct 1, oct-2A, spl, AP-2 and CTF1 (Seipel et al, EMBOJ.11, 4961-4968 (1992) and p300, CBP, PCAF, SRC1 PvALF, atHD2A and ERF-2. See, e.g., robyr et al, (2000) mol.Endocrinol.14:329-347; collingwood et al, (1999) J.mol.Endocrinol 23:255-275; leo et al, (2000) Gene 245:1-11; manteffel-Cymboowska (1999) Acta biochem.pol.46:77-89; mcKea et al, (1999) J.Steroid biochem.mol.biol.69:3-12; malik et al, (2000) Trends biochem.277-283:277; and Lemon et al, (1999) curr.Opin.Genet.Dev.9:499-504. Additional exemplary activation domains include, but are not limited to, osGAI, HALF-1, cl, AP1, ARF-5, -6, -1 and-8, CPRF1, CPRF4, MYC-RP/GP and TRAB1, see, e.g., ogawa et al, (2000) Gene 245:21-29; okanami et al, (1996) Genes Cells 1:87-99; goff et al, (1991) Genes Dev.5:298-309; cho et al, (1999) Plant Mol Biol 40:419-429; ulmason et al, (1999) Proc.Natl. Acad. Sci.USA 96:5844-5849; sprenger-Hauss et al, (2000) Plant J.22:1-8; goantJ.et al, (1999) Plant cell 1:87-99; goff et al, (1991) Genes Dev.5:298-309; cho et al, (1999) Plant Mol 40:419-429, scl.37.15, sci.37.37.

Exemplary inhibitory domains that can be used to make gene suppressors include, but are not limited to, KRAB A/B, KOX, TGF-beta-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, members of the DNMT family (e.g., DNMT1, DNMT3A, DNMT3B, DNMT L, etc.), rb, and MeCP2. See, e.g., bird et al, (1999) Cell 99:451-454; tyler et al, (1999) Cell 99:443-446; knoepflex et al, (1999) Cell 99:447-450; robertson et al, (2000) Nature Genet.25:338-342. Additional exemplary inhibitory domains include, but are not limited to, ROM2 and AtHD2A. See, for example, chem et al, (1996) Plant Cell 8:305-321; and Wu et al, (2000) Plant j.22:19-27.

In some examples, the domain relates to epigenetic regulation of the chromosome. In some embodiments, the domain is a Histone Acetyl Transferase (HAT), e.g., type a, nuclear localization such as MYST family members MOZ, ybf 2/sam 3, MOF and Tip60, GNAT family members Gcn5 or pCAF, p300 family members CBP, p300 or Rttl09 (Bemdsen and Denu (2008) Curr Opin Struct Biol (6): 682-689). In other examples, the domain is a histone deacetylase (HD AC), such as class I (HDAC-l, 2, 3 and 8), class II (HDAC IIA (HDAC-4, 5, 7 and 9), HD AC IIB (HDAC 6 and 10)), class IV (HDAC-l 1), class III (also known as Sirtuins (SIRTs); SIRT 1-7) (see Mottamal et al, (2015) Molecules 20 (3): 3898-394 l). Another domain used in some embodiments is a tissue protein phosphorylase or kinase, examples of which include MSK1, MSK2, ATR, ATM, DNA-PK, bubl, vprBP, IKK-a, PKCpi, dik/Zip, JAK2, PKC5, WSTF, and CK2. In some embodiments, a methylation domain is used and is selected from the group consisting of: such as Ezh, PRMT1/6, PRMT5/7, PRMT 2/6, CARM1, set7/9, MLL, ALL-1, suv 39h, G9a, SETDB1, ezh2, set2, dotl, PRMT1/6, PRMT5/7, PR-Set7, and Suv4-20h. Domains involved in threylation (sumoylation) and biotinylation (Lys 9, 13, 4, 18 and 12) may also be used in some embodiments (see, retrospectively, kousaride (2007) Cell 128:693-705).

Fusion molecules are constructed by cloning and biochemical conjugation methods well known to those of ordinary skill in the art to which the invention pertains. The fusion molecule comprises a DNA-binding domain and a functional domain (e.g., a transcriptional activation or inhibition domain). The fusion molecule also optionally comprises a nuclear localization signal (such as, for example, from SV40 medium T-antigen) and an epitope tag (e.g., FLAG and hemagglutinin). The fusion protein (and the encoding nucleic acid) is designed such that a translational reading frame is maintained between the fusion components.

Fusion between the polypeptide component of the functional domain (or functional fragment thereof) on the one hand and the non-protein DNA-binding domain (e.g. antibiotic, intercalator), minor groove binder, nucleic acid) on the other hand is constructed by biochemical conjugation methods known to the person skilled in the art to which the invention pertains. See, e.g., pierce Chemical Company (Rockford, IL) catalyst. Methods and compositions for making fusions between minor groove binders and polypeptides have been described. Mapp et al, (2000) Proc. Natl. Acad. Sci. USA 97:3930-3935. Likewise, CRISPR/Cas TF and nucleases (comprising an sgRNA nucleic acid component associated with a polypeptide component functional domain) are also known to those of ordinary skill in the art of the invention and are detailed herein.

Provided herein are non-activated T cells comprising reduced expression of HLA-A, HLa-B, HLA-C, CIITA, TCR- α, and/or TCR- β as compared to wild-type T cells, wherein the activated T cells further comprise a first gene encoding a Chimeric Antigen Receptor (CAR).

In some embodiments, the non-activated T cells have not been treated with an anti-CD 3 antibody, an anti-CD 28 antibody, a T cell activated cytokine, or a soluble T cell costimulatory molecule. In some embodiments, the non-activated T cells do not express an activation marker. In some embodiments, the non-activated T cells express CD3 and CD28, and wherein CD3 and/or CD28 are not activated.

In some embodiments, the anti-CD 3 antibody is OKT3. In some embodiments, the anti-CD 28 antibody is CD28.2. In some embodiments, the T cell activating cytokine is selected from the group consisting of: t cell activating cytokines composed of IL-2, IL-7, IL-15 and IL-21. In some embodiments, the soluble T cell costimulatory molecule is selected from the group consisting of: a soluble T cell costimulatory molecule consisting of an anti-CD 28 antibody, an anti-CD 80 antibody, an anti-CD 86 antibody, an anti-CD 137L antibody and an anti-ICOS-L antibody.

In some embodiments, the non-activated T cells are primary T cells. In other embodimentsNon-activated T cells differentiate from the hypoimmunity cells of the present technology. In some embodiments, the T cell is CD8 ⁺ T cells.

In some embodiments, the first gene is carried by a lentiviral vector comprising a CD8 binding agent. In some embodiments, the first gene is CAR, selected from the group consisting of: CD 19-specific CARs and CD 22-specific CARs. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the bispecific CAR is a CD19/CD22 bispecific CAR.

In some embodiments, the first and/or second gene is carried by a lentiviral vector comprising a CD8 binding agent. In some embodiments, the first and/or second genes are introduced into the cell using a fusion-mediated delivery or translocation enzyme system selected from the group consisting of: a conditional or inducible translocase, a conditional or inducible PiggyBac transposon, a conditional or inducible sleeping beauty (SB 11) transposon, a conditional or inducible Mos1 transposon and a conditional or inducible Tol2 transposon.

In some embodiments, the non-activated T cell further comprises a second gene CD47. In some embodiments, the first and/or second genes are inserted into a specific locus of at least one pair of genes of the T cell. In some embodiments, the specific locus is selected from the group consisting of: a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the second gene encoding CD47 is inserted into a specific locus selected from the group consisting of: a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the first gene encoding the CAR is inserted into a specific locus selected from the group consisting of: a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into different loci. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into the same locus. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into the B2M locus. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into the CIITA locus. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into the TRAC locus. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into the TRB locus. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into a safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of: CCR5 gene locus, CXCR4 gene locus, PPP1R12C gene locus, albumin gene locus, SHS231 gene locus, CLYBL gene locus, rosa gene locus, F3 (CD 142) gene locus, MICA gene, locus MICB gene, locus LRP1 (CD 91) gene locus, HMGB1 gene locus, ABO gene locus, RHD gene locus, FUT1 gene locus, PDGFRa gene locus, OLIG2 gene locus, GFAP gene locus and KDM5D gene locus.

In some embodiments, the non-activated T cells do not express HLA-A, HLA-B, and/or HLA-C antigens. In some embodiments, the non-activated T cells do not express B2M. In some embodiments, the non-activated T cells do not express HLA-DP, HLA-DQ, and/or HLA-DR antigens. In some embodiments, the non-activated T cells do not express CIITA. In some embodiments, the non-activated T cells do not express TCR- α and TCR- β.

In some embodiments, the non-activated T cell is B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel ^/indel A cell comprising a second gene encoding CD47 and/or a first gene encoding CAR inserted into the TRAC locus. In some embodiments, the non-activated T cell is B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel A cell comprising a second gene encoding CD47 inserted into the TRAC locus and a first gene encoding CAR. In some embodiments, the T cells are not activatedIs B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel A cell comprising a second gene encoding CD47 and/or a first gene encoding CAR inserted into a TRB locus. In some embodiments, the non-activated T cell is B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel A cell comprising a second gene encoding CD47 inserted into a TRB locus and a first gene encoding a CAR. In some embodiments, the non-activated T cell is B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel A cell comprising a second gene encoding CD47 and/or a first gene encoding CAR inserted into the B2M locus. In some embodiments, the non-activated T cell is B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel A cell comprising a second gene encoding CD47 inserted into the B2M locus and a first gene encoding CAR. In some embodiments, the non-activated T cell is B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel A cell comprising a second gene encoding CD47 and/or a first gene encoding CAR inserted into the CIITA locus. In some embodiments, the non-activated T cell is B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel/indel A cell comprising a second gene encoding CD47 inserted into the CIITA locus and a first gene encoding CAR.

Provided herein are engineered T cells comprising reduced expression of HLA-A, HLa-B, HLA-C, CIITA, TCR-a, and/or TCR- β as compared to wild-type T cells, wherein the engineered T cells further comprise a first gene encoding a Chimeric Antigen Receptor (CAR) carried by a lentiviral vector comprising a CD8 binder.

In some embodiments, the engineered T cell is a primary T cell. In other embodiments, the engineered T cells are differentiated from the hypoimmunity cells of the present technology. In some embodiments, the T cell is CD8 ⁺ T cells. In some embodiments, the T cell is CD4 ⁺ T cells.

In some embodiments, the engineered T cells do not express an activation marker. In some embodiments, the engineered T cells express CD3 and CD28, and wherein CD3 and/or CD28 are unactivated.

In some embodiments, the engineered T cells have not been treated with an anti-CD 3 antibody, an anti-CD 28 antibody, a T cell activated cytokine, or a soluble T cell costimulatory molecule. In some embodiments, the anti-CD 3 antibody is OKT3, wherein the anti-CD 28 antibody is CD28.2, wherein the T cell activating cytokine is selected from the group consisting of: a T cell activating cytokine consisting of IL-2, IL-7, IL-15 and IL-21, and wherein the soluble T cell costimulatory molecule is selected from the group consisting of: a soluble T cell costimulatory molecule consisting of an anti-CD 28 antibody, an anti-CD 80 antibody, an anti-CD 86 antibody, an anti-CD 137L antibody and an anti-ICOS-L antibody. In some embodiments, the engineered T cells have not been treated with one or more T cell activated cytokines selected from the group consisting of: IL-2, IL-7, IL-15 and IL-21. In some examples, the cytokine is IL-2. In some embodiments, one or more cytokines is IL-2 and the other is selected from the group consisting of: IL-7, IL-15 and IL-21.

In some embodiments, the engineered T cell further comprises a second gene CD47. In some embodiments, the first and/or second genes are inserted into a specific locus of at least one pair of genes of the T cell. In some embodiments, the specific locus is selected from the group consisting of: a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the second gene encoding CD47 is inserted into a specific locus selected from the group consisting of: a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the first gene encoding the CAR is inserted into a specific locus selected from the group consisting of: a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into different loci. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into the same locus. In some embodiments, the second gene encoding CD47 and the first gene encoding CAR are inserted into the B2M locus, CIITA locus, TRAC locus, TRB locus, or safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of: CCR5 gene locus, CXCR4 gene locus, PPP1R12C gene locus, albumin gene locus, SHS231 gene locus, CLYBL gene locus, rosa gene locus, F3 (CD 142) gene locus, MICA gene, locus MICB gene, locus LRP1 (CD 91) gene locus, HMGB1 gene locus, ABO gene locus, RHD gene locus, FUT1 gene locus, PDGFRa gene locus, OLIG2 gene locus, GFAP gene locus and KDM5D gene locus.

In some embodiments, the CAR is selected from the group consisting of: CD 19-specific CARs and CD 22-specific CARs.

In some embodiments, the engineered T cell does not express HLA-A, HLa-B, and/or HLa-C antigen, wherein the engineered T cell does not express B2M, wherein the engineered T cell does not express HLa-DP, HLa-DQ, and/or HLa-DR antigen, wherein the engineered T cell does not express CIITA and/or wherein the engineered T cell does not express TCR- α and TCR- β.

In some embodiments, the engineered T cell is B2M ^indel/indel 、CIITA ^indel/indel 、TRAC ^indel ^/indel The cell comprises a second gene encoding CD47 and/or a first gene encoding CAR inserted into the TRAC locus, into the TRB locus, into the B2M locus or into the CIITA locus.

In some embodiments, the non-activated T cells and/or engineered T cells of the present technology are in a subject. In other embodiments, the non-activated T cells and/or engineered T cells of the present technology are in vitro.

In some embodiments, the non-activated T cells and/or engineered T cells of the present technology express a CD8 binder. In some embodiments, the CD8 binder is an anti-CD 8 antibody. In some embodiments, the anti-CD 8 antibody is selected from the group consisting of: mouse anti-CD 8 antibodies, rabbit anti-CD 8 antibodies, human anti-CD 8 antibodies, humanized anti-CD 8 antibodies, camelid (camelid) (e.g., camelid, alpaca, camel) anti-CD 8 antibodies, and fragments thereof. In some embodiments, the fragment thereof is a scFV or VHH. In some embodiments, the CD8 binding molecule binds to the CD8 a chain and/or the CD8 β chain.

In some embodiments, the CD8 binder is fused to a transmembrane domain that is incorporated into the viral envelope. In some embodiments, the lentiviral vector is pseudotyped (pseudotyped) with a viral fusion protein. In some embodiments, the viral fusion protein comprises one or more modifications to reduce binding to its native receptor.

In some embodiments, the viral fusion protein is fused to a CD8 binder. In some embodiments, the viral fusion proteins comprise Nipah virus F glycoprotein and Nipah virus G glycoprotein fused to a CD8 binder. In some embodiments, the lentiviral vector does not comprise a T cell activating molecule or a T cell costimulatory molecule. In some embodiments, the lentiviral vector encodes a first gene and/or a second gene.

In some embodiments, the non-activated T cells or engineered T cells exhibit one or more responses selected from the group consisting of: (a) a T cell response, (b) an NK cell response, and (c) a macrophage response, which is reduced as compared to the wild-type cells after transfer to the second subject. In some embodiments, the second subject of the first subject is a different subject. In some embodiments, the macrophage response is overwhelmed.

In some embodiments, the non-activated T cells or engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in a subject, (b) reduced NK cell killing in a subject, and (c) reduced killing by whole PBMCs in a subject.

In some embodiments, the non-activated T cells or engineered T cells cause one or more selected from the group consisting of: (a) reduced donor-specific antibodies in a subject, (b) reduced IgM or IgG antibodies in a subject, and (c) reduced complement dependent cell killing (CDC) in a subject.

In some embodiments, the non-activated T cells or engineered T cells are transduced with a lentiviral vector comprising a CD8 binder in the subject. In some embodiments, the lentiviral vector carries a gene encoding CAR and/or CD 47.

Provided herein are pharmaceutical compositions comprising a population of non-activated T cells and/or engineered T cells of the present technology and a pharmaceutically acceptable additive, carrier, diluent or excipient.

Provided herein are methods comprising administering to a subject a composition comprising a population of non-activated T cells and/or engineered T cells of the present technology or one or more pharmaceutical compositions of the present technology.

In some embodiments, the subject is not administered a treatment for T cell activation before, after, and/or concurrently with administration of the composition. In some embodiments, the treatment of T cell activation comprises lymphocyte depletion (lymphodepletion).

Provided herein are methods of treating a subject having cancer comprising administering to the subject a composition comprising a population of non-activated T cells and/or engineered T cells of the present technology or one or more pharmaceutical compositions of the present technology, wherein the subject is not administered a treatment for T cell activation prior to, after, and/or concurrently with administration of the composition. In some embodiments, the treatment of T cell activation comprises lymphocyte depletion (lymphodepletion).

Provided herein are methods of expanding T cells capable of recognizing and killing tumor cells in a subject in need thereof, comprising administering to the subject a composition comprising a population of non-activated T cells and/or engineered T cells of the present technology or one or more pharmaceutical compositions of the present technology, wherein the subject is not administered a treatment for T cell activation prior to, after, and/or concurrently with administration of the composition. In some embodiments, the treatment of T cell activation comprises lymphocyte depletion (lymphodepletion).

Provided herein are dosing regimens for treating a disease or disorder in a subject comprising administering a pharmaceutical composition comprising a population of non-activated T cells and/or engineered T cells of the present technology or one or more pharmaceutical compositions of the present technology and a pharmaceutically acceptable additive, carrier, diluent or excipient, wherein the pharmaceutical composition is administered at about 1 to 3 doses.

Once altered, the presence of expression of any of the molecules described herein can be assayed using known techniques, such as western blot methods, ELISA assays, FACS assays, and the like.

Q. production of induced pluripotent Stem cells

In one aspect, provided herein are methods of producing low immunity pluripotent cells. In some embodiments, the method comprises generating pluripotent stem cells. The generation of mouse and human pluripotent stem cells (commonly referred to as iPSCs; the mouse cells are either miPSCs or the human cells are hiPSCs) is generally known in the art to which the invention pertains. Those of ordinary skill in the art will appreciate that there are a variety of different methods of generating an iPCS. The initial induction was performed by viral introduction of four transcription factors, oct3/4, sox2, c-Myc and Klf4, used from mouse embryo or adult fibroblasts; see Takahashi and Yamanaka Cell 126:663-676 (2006), incorporated herein by reference in its entirety, and in particular the techniques outlined therein. Since then, many methods have been developed; see Seki et al, world J.stem Cells 7 (1): 116-125 (2015) review, and Lakshmathy and Vermuri, editions, methods in Molecular Biology: pluripotent Stem Cells, methods and Protocols, springer 2013, which are expressly incorporated herein by reference in their entirety, and in particular methods of producing hiPSCs (see, e.g., chapter 3 of the latter reference).

Generally, the iPSC gene is produced by transient expression of one or more reprogramming factors "in the host cell, typically introduced using episomal vectors. Under these conditions, a small number of cells were induced to iPSC (generally, this step was inefficient because no selection marker was used). Once cells are "reprogrammed" and made pluripotent, they lose episomal vectors and use endogenous genes to produce factors.

The number of reprogramming factors that may be used or employed may vary, as will be appreciated by those of ordinary skill in the art to which the invention pertains. In general, the efficiency of cells to transform into a pluripotent state decreases when fewer reprogramming factors are used, and "pluripotency", e.g., fewer reprogramming factors may result in cells that are not fully pluripotent but may only be able to differentiate into fewer cell types.

In some embodiments, OCT4 is used with a single reprogramming (reprogramming) factor. In other embodiments, two reprogramming (reprogramming) factors are used, OCT4 and KLF4. In other embodiments, three reprogramming factors, OCT4, KLF4, and SOX2, are used. In other embodiments, four reprogramming factors are used, OCT4, KLF4, SOX2, and c-Myc. In other embodiments, 5, 6, or 7 reprogramming (reprogramming) factors selected from the following may be used: SOKMNLT; SOX2, OCT4 (POU 5F 1), KLF4, MYC, NANOG, LIN, and SV40L T antigens. In general, these reprogramming factor genes are provided to episomal vectors, such as are known and commercially available in the art to which the invention pertains.

Generally, ipscs are made from non-pluripotent cells, such as, but not limited to, blood cells, fibroblasts, etc., by transiently expressing reprogramming factors as described herein, as is known in the art.

Assays for retention of low immunity phenotype and pluripotency

Once the hypoimmune cells have been generated, they may be assayed for retention of their hypoimmunity and/or pluripotency, as described in WO2016183041 and WO2018132783.

In some embodiments, the low immunity is assayed using a variety of techniques exemplified in figures 13 and 15 of WO2018132783. These techniques include transplantation into an allogeneic host, and monitoring low immunity pluripotent cell growth (e.g., teratomas) that escape the host's immune system. In some examples, the low immunity pluripotent cell derivative is transduced to express luciferase and can then be tracked using bioluminescence imaging. Likewise, T cells and/or B cells of the host animal are tested for response to such cells to confirm that the cells do not elicit an immune response in the host animal. T cell responses can be assessed by Elispot, ELISA, FACS, PCR or mass flow Cytometry (CYTOF). B cell responses or antibody responses were assessed using FACS or Luminex. Additionally or alternatively, cells may be assayed for their ability to avoid an innate immune response, e.g., NK cell killing, as generally shown in figures 14 and 15 of WO2018132783.

In some embodiments, cellular immunity is assessed by T cell immunoassays, such as T cell proliferation assays, T cell activation assays, and T cell killing assays, that are recognized by those of ordinary skill in the art of the invention. In some examples, the T cell proliferation assay comprises pre-treating cells with interferon-gamma and co-culturing cells with labeled T cells, and assaying for the presence of a T cell population (or a proliferated T cell population) after a preselected amount of time. In some examples, the T cell activation assay comprises co-culturing T cells with cells as outlined herein and determining the amount of expression of the T cell activation marker in the T cells.

In vivo assays can be performed to assess the immunity of the cells outlined herein. In some embodiments, the survival and immunity of the hypoimmunity cells is determined using an allogeneic humanized immunodeficiency mouse model. In some examples, low immunity pluripotent stem cells are transplanted into allogeneic humanized NSG-SGM3 mice and assayed for cell rejection, cell survival, and teratoma formation. In some examples, the transplanted low immunity pluripotent stem cells or differentiated cells thereof exhibit long term survival in a mouse model.

Other techniques for determining immunity, including low immunity of cells, are described, for example, in Deuse et al, nature Biotechnology,2019,37,252-258 and Han et al, proc Natl Acad Sci USA,2019,116 (21), 10441-10446, the disclosures of which, including descriptions of the drawings, graphic legends and methods, are incorporated herein by reference in their entirety.

Likewise, retention of pluripotency is tested in a variety of ways. In one embodiment, pluripotency is assayed by expression of certain pluripotency-specific factors as generally described herein and shown in figure 29 of WO 2018132783. Additionally or alternatively, differentiation of pluripotent cells into one or more cell types is indicative of pluripotency.

As will be appreciated by those of ordinary skill in the art to which the invention pertains, successful reduction of MHC I function (HLA I, when the cells are derived from human cells) in pluripotent cells can be measured using techniques known in the art to which the invention pertains and described below; for example, FACS techniques using labeled antibodies that bind to HLA complexes; for example, use is made of commercially available HLA-A, B, C antibodies (alpha chains binding to human major histocompatibility HLa class I antigens).

In addition, cells can be tested to confirm that HLA I complexes are not expressed on the cell surface. This can be done by FACS analysis, using an antibody assay against one or more of the HLA cell surface components discussed above.

Successful reduction of MHC II function (HLA II, when the cells are derived from human cells) in pluripotent cells or derivatives thereof can be measured using techniques known in the art to which the invention pertains, such as western blot methods using antibodies to proteins, FACS techniques, RT-PCR techniques, and the like.

In addition, cells can be tested to confirm that HLA II complexes are not expressed on the cell surface. Again, assays are performed as known in the art to which the invention pertains (see, e.g., fig. 21 of WO 2018132783) and are generally done using western blot or FACS analysis based on commercial antibodies that bind to human HLA class II HLA-DR, DP and most DQ antigens.

In addition to the reduction of HLA I and II (or MHC I and II), the low immunity cells provided herein have reduced susceptibility to macrophage phagocytosis and NK cell killing. The resulting low-immunity cells "escape" immune macrophages and innate pathways due to the expression of one or more CD24 transgenes.

S. maintenance of pluripotent Stem cells

Once the low immunity pluripotent stem cells are generated, they can remain in an undifferentiated state, as is known to maintain ipscs. For example, cells are cultured on Matrigel using a medium to avoid differentiation and maintain pluripotency. In addition, it may be in a medium under conditions that maintain pluripotency.

T. differentiated cells from low immunity induced pluripotent (HIP) stem cells

In one aspect, provided herein are HIP cells that differentiate into different cell types for subsequent transplantation into a recipient subject. Differentiation can be assayed as known in the art, typically by assessing the presence of cell-specific markers. As will be appreciated by those of ordinary skill in the art, differentiated low immunity multipotent cell derivatives can be transplanted using techniques known in the art of the invention, depending on the cell type and end use of these cells.

1. Cardiac cells differentiated from low immunity pluripotent cells

Provided herein are cardiac cell types differentiated from HIP cells for subsequent transplantation or implantation into a subject (e.g., recipient). As will be appreciated by those of ordinary skill in the art to which the invention pertains, the method of differentiation depends on the desired cell type using known techniques. Exemplary cardiac cell types include, but are not limited to, cardiomyocytes, nodular cardiomyocytes, conducting cardiomyocytes, working cardiomyocytes, cardiomyocyte precursor cells, cardiomyocyte progenitor cells, cardiac stem cells, cardiac muscle cells, atrial cardiac stem cells, ventricular cardiac stem cells, epicardial cells, hematopoietic cells, vascular endothelial cells, endocardial endothelial cells, cardiac valve stromal cells, cardiac pacing cells, and the like.

In some embodiments, the cardiac cells described herein are administered to a recipient subject to treat a cardiac disorder selected from the group consisting of: pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, localized cardiomyopathy, chronic ischemic cardiomyopathy, pre-and post-partum cardiomyopathy, inflammatory cardiomyopathy, spontaneous cardiomyopathy, other cardiomyopathy, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end-stage heart disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, cardiovascular disease, myocardial infarction, myocardial ischemia, congestive heart failure, myocardial infarction, cardiac ischemia, cardiac injury, myocardial ischemia, vascular disease, acquired heart disease, congenital heart disease, atherosclerosis, coronary artery disease, abnormal functional conduction system, abnormal coronary artery, pulmonary hypertension, cardiac arrhythmia, muscular dystrophy, abnormal muscle mass, muscle degeneration, myocarditis, infectious myocarditis, drug-or toxin-induced muscular abnormality, allergic myocarditis, and autoimmune endocarditis.

Accordingly, provided herein are methods for treating and preventing heart injury or heart disease or disorder in a subject in need thereof. The methods described herein can be used to treat, ameliorate, prevent or slow the progression of a number of heart diseases or symptoms thereof, such as those that cause pathological damage to the structure and/or function of the heart and brain. The terms "heart disease," "heart disorder," and "heart injury" are used interchangeably herein and refer to conditions and/or disorders associated with the heart, including valves, endothelium, infarct zone, or other components or structures of the heart. Such heart diseases or heart related diseases include, but are not limited to, myocardial infarction, heart failure, cardiomyopathy, congenital heart defects, heart valve diseases or dysfunction, endocarditis, rheumatic fever, mitral valve prolapse, infectious endocarditis, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis, enlarged heart, and/or mitral valve insufficiency, among others.

In some embodiments, the cardiomyocyte precursor comprises cells capable of producing progeny, including mature (end-stage) cardiomyocytes. Cardiomyocyte precursor cells can be generally identified using one or more markers selected from the MEF-2 family of GATA-4, nkx2.5 and transcription factors. In some examples, cardiomyocytes are immature cardiomyocytes or expression is derived from List of one or more markers (sometimes at least 2, 3, 4 or 5 markers) of mature cardiomyocytes: cardiac troponin I (cTnl), cardiac troponin T (cTnt), myofibrillar segments (sarcomeric) Myosin Heavy Chain (MHC), GATA-4, nkx2.5, N-adhesion protein, beta 2-adrenoceptor, ANF, MEF-2 family of transcription factors, creatine kinase MB (CK-MB), myoglobin and Atrial Natriuretic Factor (ANF). In some examples, when combined with appropriate Ca in an appropriate tissue culture environment ²⁺ When heart cells are cultured with concentration and electrolyte balance, it is observed that the cells contract in a periodic fashion on one axis of the cells and then release from the contraction without having to add additional components to the medium. In some embodiments, the cardiac cell is a low immunity cardiac cell.

In some embodiments, a method of generating a population of low immunity heart cells from a population of low immunity pluripotent (HIP) cells by in vitro differentiation comprises: (a) Culturing a population of HIP cells in a medium comprising a GSK inhibitor; the method comprises the steps of carrying out a first treatment on the surface of the (b) Culturing a population of HIP cells in a medium comprising a WNT antagonist to produce a population of precordial cells; and (c) culturing the population of precordial cells in a medium comprising insulin to produce a population of hyperimmune heart cells. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some examples, the concentration of GSK inhibitor ranges from about 2mM to about 10mM. In some embodiments, the WNT antagonist is IWR1, a derivative or variant thereof. In some examples, the concentration of WNT antagonist ranges from about 2mM to about 10mM.

In some embodiments, a population of low immunity cardiac cells is isolated from non-cardiac cells. In some embodiments, an isolated population of low immunity cardiac cells is expanded prior to administration. In certain embodiments, an isolated population of low immunity heart cells is expanded and cryopreserved prior to administration.

In some embodiments, the pluripotent cells differentiate into cardiomyocytes to address cardiovascular disease. Techniques for differentiating hipscs into cardiomyocytes are known in the art to which the invention pertains and are discussed in embodiments. Differentiation can be assayed as known in the art, typically by assessing the presence of cardiomyocyte-associated or specific markers or by functional measurement; see, e.g., loh et al, cell,2016,166,451-467, incorporated herein by reference in its entirety, particularly for methods of differentiating stem cells, including cardiomyocytes.

Other useful methods for differentiating induced pluripotent stem cells or pluripotent stem cells into cardiac cells are described, for example, in US2017/0152485; US2017/0058263; US2017/0002325; US2016/0362661; US2016/0068814; US9,062,289; US7,897,389; and US7,452,718. Additional methods for generating cardiac cells from induced pluripotent Stem cells or multipotent Stem cells are described, for example, in Xu et al Stem Cells and Development,2006,15 (5): 631-9, burridge et al, cell Stem Cell 2012,10:16-28 and Chen et al, stem Cell Res,2015,15 (2): 365-375.

In various embodiments, the hypoimmune cardiac cells can be cultured in a medium comprising BMP pathway inhibitors, WNT signaling activators, WNT signaling inhibitors, WNT agonists, WNT antagonists, src inhibitors, EGFR inhibitors, PCK activators, cytokines, growth factors, cardiophilic agents, compounds, and the like.

WNT signaling activators include, but are not limited to, CHIR99021.PCK activators include, but are not limited to, PMA. WNT signaling inhibitors include, but are not limited to, compounds selected from KY02111, SO3031 (KY 01-I), SO2031 (KY 02-I) and SO3042 (KY 03-I) and XAV939. Src inhibitors include, but are not limited to, a419259.EGFR inhibitors include, but are not limited to AG1478.

Non-limiting examples of agents that produce cardiac cells from ipscs include activin A, BMP, wnt3a, VEGF, soluble frizzled proteins, cyclosporin a, angiotensins II, phenylephrine, ascorbic acid, dimethyl sulfoxide, 5-aza-2' -deoxycytidine, and the like.

The cells provided herein can be cultured on a surface, such as a synthetic surface, to support and/or promote differentiation of low immunity pluripotent cells into cardiac cells. In some embodiments, the surface comprises a polymeric material including, but not limited to, a homopolymer or copolymer selected from one or more acrylate monomers. Acrylic ester monomer and A Non-limiting examples of the methacrylate monomers include tetra (ethylene glycol) diacrylate, glycerol dimethacrylate, 1, 4-butanediol dimethacrylate, poly (ethylene glycol) diacrylate, di (ethylene glycol) dimethacrylate, tetra (ethylene glycol) dimethacrylate, 1, 6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, trimethylolpropane ethoxylate (1 EO/QH) methyl, tricyclo [5.2.1.0 ] ^2,6 ]Decanedimethanol diacrylate, neopentyl glycol ethoxylate diacrylate and trimethylolpropane triacrylate. Synthetic acrylates are known in the art of the invention or are available from commercial suppliers such as Polysciences, inc., sigma Aldrich, inc.

The polymeric material may be dispersed on the surface of the support material. Useful support materials suitable for culturing cells include ceramic substances, glass, plastics, polymers or copolymers, any combination thereof, or coatings of one material on another. In some examples, the glass includes soda lime glass, boron glass, silica glass, quartz glass, silicon or derivatives of these, and the like.

In some examples, the plastic or polymer comprising the dendritic polymer comprises poly (vinyl chloride), poly (vinyl alcohol), poly (methyl methacrylate), poly (vinyl acetate-maleic anhydride), poly (dimethylsiloxane) monomethacrylate, cyclic olefin polymer, fluorocarbon polymer, polystyrene, polypropylene, polyethyleneimine or derivatives of these, and the like. In some examples, the copolymer includes poly (vinyl acetate-co-maleic anhydride), poly (styrene-co-maleic anhydride), poly (ethylene-co-acrylic acid), derivatives of these, or the like.

The efficacy of heart cells prepared as described herein can be assessed in animal models of cardiac freeze injury (resulting in 55% of left ventricular wall tissue becoming sCAR-tissue without treatment) (Li et al, ann. Thorac. Surg.62:654,1996; sakai et al, ann. Thorac. Surg.8:2074,1999, sakai et al, thorac. Cardiovasc. Surg.118:715,1999). Successful treatment may reduce scar area, limit scar dilation, and improve cardiac function as determined by systolic, diastolic, and developing pressures. Embolic coils can also be used at the distal portion of the left anterior descending branch artery to establish patterned cardiac injury (Watanabe et al, cell transition.7: 239,1998), and therapeutic efficacy can be assessed by histology and cardiac function.

In some embodiments, administering includes implanting cardiac tissue, intravenous injection, intra-arterial injection, intra-coronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, endocardial injection, epicardial injection, or infusion into a tissue subject.

In some embodiments, the patient administered the engineered cardiac cells is also administered a cardiac drug. Illustrative examples of cardiac drugs suitable for use in combination therapy include, but are not limited to, growth factors, polynucleotides encoding growth factors, angiogenic agents, calcium channel blockers, antihypertensives, antimitotics, cardiotonic agents, anti-atherosclerosis agents, anticoagulants, beta-blockers, antiarrhythmic agents, anti-inflammatory agents, vasodilators, thrombolytic agents, cardiac glycosides, antibiotics, antiviral agents, antifungal agents, protozoan inhibiting agents, nitrates, angiotensin Converting Enzyme (ACE) inhibitors, angiotensin ii receptor antagonists, brain Natriuretic Peptide (BNP); antitumor agents, steroids, etc.

The effectiveness of therapy according to the methods provided herein can be monitored in a variety of ways. For example, an Electrocardiogram (ECG) or holier monitor may be used to determine treatment efficacy. ECG is a measurement of cardiac rhythms and electrical impulses, and is a very effective and non-invasive method to determine if therapy improves or maintains, prevents or slows electrical conduction degradation in the subject's heart. Portable ECG using a holier monitor, wearable over a long period of time to monitor cardiac abnormalities, arrhythmia disorders, and the like, is also a reliable method of assessing the effectiveness of therapy. ECG or nuclear studies can be used to determine improvement in ventricular function.

2. Neural cells differentiated from low immunity pluripotent cells

Provided herein are different neural cell types differentiated from HIP cells useful for subsequent transplantation or implantation into a recipient subject. As will be appreciated by those of ordinary skill in the art to which the invention pertains, the method of differentiation depends on the desired cell type using known techniques. Exemplary neural cell types include, but are not limited to, brain endothelial cells, neurons (e.g., dopamine neurons), glial cells, and the like.

In some embodiments, differentiation of induced pluripotent stem cells is performed by exposing or contacting the cells to specific factors known to produce a specific cell lineage, thereby targeting their differentiation to a specific, desired lineage and/or cell type of interest. In some embodiments, the terminally differentiated cells exhibit a specific phenotypic characteristic or characteristic. In certain embodiments, the stem cells described herein differentiate into a neuroectodermal, neuronal, neuroendocrine, dopamine, biliary, serotonergic (5-HT), glutamate, GABAergic, adrenergic, noradrenergic, sympathetic, parasympathetic, sympathetic peripheral, or glial cell population. In some examples, the population of glial cells includes a population of microglia (e.g., amoeboid, divergent, active phagocytic, and active non-phagocytic) cells or megaly (central nervous system cells: astrocytes, oligodendrocytes, ependymal cells, and radial glial cells; and peripheral nervous system cells: schwann cells and satellite cells) cells or precursors and progenitors of any of the foregoing.

PCT application No. WO2010144696, U.S. patent No. 9,057,053;9,376,664; and protocols for producing different types of neural cells described in 10,233,422. Additional description of methods of differentiating low immunity pluripotent cells can be found, for example, in Deuse et al, nature Biotechnology,2019,37,252-258 and Han et al, proc Natl Acad Sci USA,2019,116 (21), 10441-10446. Methods for determining the effects of neural cell transplantation in animal models of neurological disorders or conditions are described in the following references: for spinal cord injury-Curtis et al, cell Stem Cell,2018,22,941-950; for Parkinson's disease-Kikuchi et al, nature,2017,548:592-596; for ALS-Izrael et al Stem Cell Research,2018,9 (1): 152 and Izrael et al, interchOpen, DOI: 10.5772/intelhopen.72862; for epilepsy -Uppaphya et al, PNAS,2019,116 (1): 287-296.

a. Brain endothelial cells

In some embodiments, the neural cells are administered to a subject to treat parkinson's disease, huntington's disease, multiple sclerosis, other neurodegenerative diseases or disorders, attention Deficit Hyperactivity Disorder (ADHD), tourette's disease (TS), dysesthesia, psychosis, depression, other neuropsychiatric disorders. In some embodiments, the neural cells described herein are administered to a subject to treat or ameliorate stroke. In some embodiments, neurons and glial cells are administered to subjects with Amyotrophic Lateral Sclerosis (ALS). In some embodiments, brain endothelial cells are administered to alleviate symptoms or effects of cerebral hemorrhage. In some embodiments, the dopamine neuron is administered to a patient suffering from parkinson's disease. In some embodiments, the noradrenergic neurons, GABAergic interneurons, are administered to patients experiencing seizures. In some embodiments, motor neurons, interneurons, schwann cells, oligodendrocytes, and microglia are administered to a patient experiencing spinal cord injury.

In some embodiments, brain Endothelial Cells (ECs), precursors and precursors thereof are differentiated from surface pluripotent stem cells (e.g., induced pluripotent stem cells) by culturing the cells in a medium comprising one or more factors that promote the production of brain ECs or nerve cells. In some examples, the medium includes one or more of the following: CHIR-99021, VEGF, basic FGF (bFGF) and Y-27632. In some embodiments, the culture medium includes supplements designed to promote neural cell survival and functionality.

In some embodiments, brain Endothelial Cells (ECs), precursors and precursors thereof are differentiated from pluripotent stem cells at the surface by culturing the cells in an unregulated or conditioned medium. In some examples, the medium comprises factors or small molecules that promote or assist in differentiation. In some embodiments, the culture medium comprises one or more factors or small molecules selected from the group consisting of: VEGR, FGF, SDF-1, CHIR-99021, Y-27632, SB 431542 and combinations thereof. In some embodiments, the surface for differentiation comprises one or more extracellular matrix proteins. The surface may be coated with one or more extracellular matrix proteins. Cells can be differentiated in suspension and then placed into a gel matrix form, such as matrigel, gelatin, or fibrin/thrombin form, to assist in cell survival. In some examples, differentiation is generally assayed by assessing the presence of cell-specific markers, as is known in the art to which the invention pertains.

In some embodiments, the brain endothelial cells express or secrete factors selected from the group consisting of: CD31, VE adhesion proteins, and combinations thereof. In certain embodiments, the brain endothelial cells express or secrete one or more factors selected from the group consisting of: CD31, CD34, CD45, CD117 (c-kit), CD146, CXCR4, VEGF, SDF-1, PDGF, GLUT-1, PECAM-1, eNOS, fibronectin-5, clusterin, ZO-1, p-glycoprotein, feng Wei Rich (von Willebrand) factor, VE-adhesion protein, low density lipoprotein receptor LDLR, low density lipoprotein receptor-related protein 1LRP1, insulin receptor INSR, leptin receptor LEPR, basal cell adhesion molecule BCAM, transferrin receptor TFRC, advanced glycation end product-specific receptor AGER, receptor for retinol uptake STRA6, large neutral amino acid transporter small subunit 1SLC7A5, excitatory amino acid transporter 3SLC1A1, sodium-coupled neutral amino acid transporter 5SLC38A5, carrier family 16 member 1SLC16A1, ATP-dependent translocase CB1, ATP-CC 2-binding cassette transporter AB2, late glycosylation end product-specific receptor AGER, excitatory amino acid transporter 3SLC1A1, and multi-protein-related drug-2-receptor ABCC2, multi-protein-related drug-3, multi-protein-specific drug-2 ABCC2, and multi-drug-related drug-4-protein-ABC 2.

In some embodiments, the brain EC is characterized by one or more characteristics selected from the group consisting of: tightly linked high expression, high resistance, low fenestration, small perivascular space, high transmission of insulin and transferrin receptor, and high mitochondrial numbers.

In some embodiments, a positive selection strategy is used to select or purify brain ECs. In some examples, brain ECs are sorted for endothelial cell markers, such as, but not limited to, CD31. In other words, CD31 positive brain ECs were isolated. In some embodiments, brain ECs are selected or purified using a negative selection strategy. In some embodiments, undifferentiated or pluripotent stem cells are removed by selecting cells expressing a pluripotency marker (including, but not limited to, TRA-1-60 and SSEA-1).

b. Dopamine neurons

In some embodiments, HIP cells described herein differentiate into dopamine neurons, including neuronal stem cells, neuronal progenitor cells, immature dopamine neurons, and mature dopamine neurons.

In some examples, the term "dopamine neuron" includes a neuronal cell that expresses Tyrosine Hydroxylase (TH), the rate-limiting enzyme of dopamine synthesis. In some embodiments, the dopamine neurons secrete neurotransmission dopamine, and little or no expression of dopamine hydroxylase is present. Dopamine (DA) neurons can express one or more of the following markers: neuronal-specific enolase (NSE), 1-aromatic amino acid decarboxylase, vesicle monoamine transporter 2, dopamine transporter, nurr-l and dopamine-2 receptor (D2 receptor). In certain examples, the term "neural stem cell" includes a population of pluripotent cells that have been partially differentiated along a neural cell pathway and that express one or more neural markers, including, for example, nestin (nestin). Neural stem cells can differentiate into neurons or glial cells (e.g., astrocytes and oligodendrocytes). The term "neural progenitor cells" includes cultured cells that express FOXA2 and low amounts of b-tubulin, but are free of tyrosine hydroxylase. This neural progenitor cell has the ability to differentiate into various neuronal subtypes; in particular, various subtypes of dopamine neurons, such as those described herein, after culturing the appropriate factors.

In some embodiments, DA neurons derived from HIP cells are administered to a patient, e.g., a human patient, to treat a neurodegenerative disease or disorder. In some examples, the neurodegenerative disorder or disease is selected from the group consisting of: parkinson's disease, huntington's disease, and multiple sclerosis. In other embodiments, the DA neurons are used to treat or ameliorate one or more symptoms of a neuropsychiatric disorder, such as Attention Deficit Hyperactivity Disorder (ADHD), tourette's disease (TS), dysesthesia, psychosis, and depression. In yet other embodiments, the DA neurons are used to treat patients with impaired DA neurons.

In some embodiments, the DA neurons, precursors and precursors thereof are differentiated from pluripotent stem cells by culturing the stem cells in a medium comprising one or more factors or additives. Useful factors and additives that promote differentiation, growth, expansion, maintenance and/or maturation of DA neurons include, but are not limited to, wntl, FGF2, FGF8a, sonic hedgehog (SHH), brain-derived neurotrophic factor (BDNF), transforming growth factor a (TGF-a), TGF-B, metabain 1 beta, glial cell line-derived neurotrophic factor (GDNF), GSK-3 inhibitor (e.g., CHIR-99021), TGF-B inhibitor (e.g., SB-431542), B-27 supplement, doxoform (dorsomorphin), pummorphomine (noggin), retinoic acid, cAMP, ascorbic acid, neurorank protein (neurturin), knock-out serum replacement, N-acetyl cysteine, c-kit ligand, modified forms thereof, mimetics, analogs thereof, and variants thereof. In some embodiments, the DA neurons differentiate in the presence of one or more factors that activate or inhibit WNT pathway, NOTCH pathway, SHH pathway, BMP pathway, FGF pathway, and the like. Differentiation procedures and detailed descriptions thereof are provided, for example, in US9,968,637, US7,674,620, kim et al, nature,2002,418,50-56; bjorklund et al, PNAS,2002,99 (4), 2344-2349; grow et al Stem Cells Transl Med.2016,5 (9): 1133-44 and Cho et al PNAS 2008,105:3392-3397, the entire disclosure including detailed descriptions of embodiments, methods, drawings and results are incorporated herein by reference.

In some embodiments, a population of hypoimmunity dopamine neurons is isolated from non-neuronal cells. In some embodiments, an isolated population of low-immunity dopamine neurons is amplified prior to administration. In certain embodiments, an isolated population of hypo-immune dopamine neurons is amplified and cryopreserved prior to administration.

To characterize and monitor DA formation and assess DA phenotype, the expression of any number of molecules and gene markers can be assessed. For example, the presence of a genetic marker may be determined by various methods known to those of ordinary skill in the art to which the invention pertains. Expression of the molecular markers may be determined by quantitative methods such as, but not limited to, qPCR-based assays, immunoassays, immunocytochemical assays, immunoblots assays, and the like. Exemplary markers for DA neurons include, but are not limited to, TH, B-tubulin, pax6, insulin gene enhancer protein (Isl 1), nestin (nestin), diaminobenzidine (DAB), G-protein activated inward rectifying Potassium channel 2 (GIRK 2), microtubule-associated protein 2 (MAP-2), NURR1, dopamine transporter (DAT), fork-box protein A2 (FOXA 2), FOX3, bisdermatan (daulecortin), LIM homeobox transcription factor l-beta (LMX 1B), and the like. In some embodiments, the DA neuron expresses one or more markers selected from the group consisting of: corin, FOXA2, tuJ1, NURR1, and combinations thereof.

In some embodiments, the DA neurons are assessed according to cellular electrophysiological activity. The electrophysiology of cells can be assessed by using assays known to those of ordinary skill in the art to which the invention pertains. Such as whole cell and perforated patch clamp, assays to detect cell electrophysiological activity, assays to measure the magnitude and duration of cell action potentials, and functional assays to detect dopamine production by DA cells.

In some embodiments, DA neuron differentiation is characterized by spontaneous rhythmic action potentials and high frequency action potentials with spike frequency adaptation upon injection of depolarization currents. In other embodiments, the DA differentiation is characterized by dopamine production. The amount of dopamine produced is calculated by measuring the width of the action potential at the point when it reaches half its maximum amplitude (spike half-maximum width).

In some embodiments, the differentiated DA neurons are transplanted intravenously or by injection into a specific location in a patient. In some embodiments, the differentiated DA cells are transplanted into the substantia nigra of the brain (particularly in the dense region or adjacent regions), ventral cap membrane region (VTA), caudate nucleus, putamen, metacarpal, subvisual nucleus (subthalamic nucleus), or any combination thereof, in place of the degenerated DA neurons that cause parkinson's disease. Differentiated DA cells may be injected as a cell suspension into the target area. Alternatively, the differentiated DA cells may be embedded in a supporting matrix or scaffold when included in such a delivery device. In some embodiments, the scaffold is biodegradable. In other embodiments, the scaffold is non-biodegradable. The scaffold may comprise natural or synthetic (artificial) materials.

Delivery of the DA neurons may be achieved through the use of suitable vehicles such as, but not limited to, liposomes, microparticles, or microcapsules. In other embodiments, the differentiated DA neurons are administered in a pharmaceutical composition comprising isotonic excipients. The pharmaceutical composition is prepared under conditions sufficiently sterile for human administration. In some embodiments, the DA neurons differentiated from HIP cells are provided in the form of a pharmaceutical composition. General principles for therapeutic formulation of Cell compositions can be found in Cell therapy: stem Cell Transplantation, gene Therapy, and Cellular Immunotherapy, G.Morstyn & W.Shredan eds, cambridge University Press,1996 and Hematopoietic Stem Cell Therapy, E.Ball, J.Lister & P.Law, churchill Livingstone,2000, the disclosures of which are incorporated herein by reference.

Useful descriptions of stem Cell-derived neurons and methods of their manufacture can be found, for example, in Kirkeby et al, cell Rep,2012,1:703-714; kriks et al, nature,2011,480:547-551; wang et al Stem Cell Reports,2018,11 (1): 171-182; lorenz studio, "Chapter 8-Strategies for Bringing Stem Cell-Derived Dopamine Neurons to The clinic-The NYSTEM Trial" in Progress in Brain Research,2017,volume 230,pg.191-212; liu et al, nat Protoc,2013,8:1670-1679; upadhea et al Curr Protoc Stem Cell Biol,38,2d.7.1-2d.7.47; US patent publication No. 20160115448 and US8,252,586; US8,273,570; US9,487,752 and US10,093,897 find that the contents of which are incorporated herein by reference in their entirety.

In addition to DA neurons, other neuronal cells, precursors and precursors thereof can be differentiated from HIP cells as outlined herein by culturing the cells in a medium comprising one or more factors or additives. Non-limiting examples of factors and additives include GDNF, BDNF, GM-CSF, B27, basic FGF, basic EGF, NGF, CNTF, SMAD inhibitor, wnt antagonist, SHH signaling activator, and combinations thereof. In some embodiments, the SMAD inhibitor is selected from the group consisting of: SB431542, LDN-193189, noggin (Noggin) PD169316, SB203580, LY364947, A77-01, A-83-01, BMP4, GW788388, GW6604, SB-505124, lepidmumab, met-mumab, GC-I008, AP-12009, AP-110I4, LY550410, LY580276, LY364947, LY2109761, SB-505124, E-616452 (RepSox ALK inhibitor), SD-208, SMI6, NPC-30345, K26894, SB-203580, SD-093, activin-M108A, P144, soluble TBR2-Fc, DMH-1, doxofmorphine dihydrochloride and derivatives thereof. In some embodiments, the Wnt antagonist is selected from the group consisting of: XAV939, DKK1, DKK-2, DKK-3, DKK-4, SFRP-1, SFRP-2, SFRP-3, SFRP-4, SFRP-5, WIF-1, soggy, IWP-2, IWR1, ICG-001, KY0211, wnt-059, LGK974, IWP-L6 and derivatives thereof. In some embodiments, the SHH signaling activator is selected from the group consisting of: s Mo Sende (Smoothened) agonists (SAG), SAG analogues, SHH, C25-SHH, C24-SHH, puminorphine (purporthamine), hg-Ag and/or derivatives thereof.

In some embodiments, the neuron expresses one or more markers selected from the group consisting of: glutamate ion receptor NMDA type subunit 1GRIN1, glutamate decarboxylase 1GAD1, gamma-aminobutyric acid GABA, tyrosine hydroxylase TH, LIM homeobox transcription factor 1-alpha LMX1A, fork box protein O1 FOXO1, fork box protein A2 FOXA2, fork box protein O4 FOXO4, FOXG1, 2',3' -cyclic-nucleotide 3' -phosphodiesterase CNP, myelin basic protein MBP, tubulin beta chain 3TUB3, tubulin beta chain 3NEUN, solute carrier family 1 member 6SLC1A6, SST, PV, calbindin (calbindin), RAX, LHX6, LHX8, DLX1, DLX2, DLX5, DLX6, SOX6, MAFB, NPAS1, ASCL1, SIX6, OLIG2, NKX2.1, NKX2.2, NKX6.2, VGLUT1, MAP2, CTIP2, SATB2, TBR1, DLX2, ASCL1, chAT, NGFI-B, c-fos, CRF, RAX, POMC, hypocretin, NADPH, NGF, ach, VAChT, PAX6, EMX2p75, CORIN, TUJ1, NURR1, and/or any combination thereof.

c. Glial cells

In some embodiments, the neural cells include glial cells, such as, but not limited to, microglia, astrocytes, oligodendrocytes, ependymal cells and schwann cells, their glial precursors and glial precursors are produced by differentiating pluripotent stem cells into therapeutically effective glial cells, and the like. Differentiation of the low-immunity pluripotent stem cells produces low-immunity neural cells, such as low-immunity glial cells.

In some embodiments, the glial cells, precursors and precursors thereof are produced by culturing pluripotent stem cells in a medium comprising one or more agents selected from the group consisting of: retinoic acid, IL-34, M-CSF, FLT3 ligand, GM-CSF, CCL2, TGF beta inhibitor, a BMP signaling inhibitor, SHH signaling activator, FGF, platelet derived growth factor PDGF, PDGFR-alpha, HGF, IGF1, noggin, SHH, doxoform, noggin, and combinations thereof. In some examples, the BMP signaling inhibitor is LDN193189, SB431542, or a combination thereof. In some embodiments, the glial cells express NKX2.2, PAX6, SOX10, brain-derived neurotrophic factor BDNF, neurotrophin (neutrotrophin) -3NT-3, NT-4, EGF, ciliary neurotrophic factor CNTF, nerve growth factor NGF, FGF8, EGFR, OLIG1, OLIG2, myelin basic protein MBP, GAP-43, LNGFR, nestin (nestin), GFAP, CD11b, CD11c, CX3CR1, P2RY12, IBA-1, TMEM119, CD45, and combinations thereof. Exemplary differentiation media may include any specific factor and/or small molecule recognized by those of ordinary skill in the art to assist or promote glial cell type production.

To determine whether cells generated according to in vitro differentiation procedures exhibit glial cell characteristics and features, cells can be transplanted into animal models. In some embodiments, the glial cells are injected into immunocompromised mice, e.g., a shaky mouse that is immunocompromised. Glial cells were applied to the brain of mice and after a preselected amount of time, the implanted cells were evaluated. In some examples, the implanted cells in the brain are visualized by immunostaining and imaging methods. In some embodiments, glial cell expression is determined as a known glial cell biomarker.

Useful methods for generating glial cells, precursors and precursors thereof from stem cells are for example, US7,579,188; US7,595,194; US8,263,402; US8,206,699; US8,252,586; US9,193,951; US9,862,925; US8,227,247; US9,709,553; US2018/0187148; US2017/0198255; US2017/0183627; US2017/0182097; US2017/253856; US 2018/023604; WO2017/172976; and WO 2018/093681. Methods of differentiating pluripotent stem cells are described, for example, in Kikuchi et al, nature,2017,548,592-596; kriks et al Nature,2011,547-551; doi et al Stem Cell Reports,2014,2,337-50; perrier et al Proc Natl Acad Sci USA,2004,101,12543-12548; chambers et al, nat Biotechnol,2009,27,275-280; and Kirkeby et al, cell reports, 2012,1,703-714.

The efficacy of nerve cell grafts on spinal cord injury can be found, for example, in McDonald, et al, nat.med.,1999,5:1410 Kim, et al, nature,2002,418): 50 in an acute impaired spinal cord rat model. For example, a successful graft may show that graft-derived cells are present at the foci, e.g., differentiate into astrocytes, oligodendrocytes and/or neurons, and migrate from the focal end along the spinal cord after 2 to 5 weeks, and improve gait, coordination and loading. A specific animal model is selected based on the type of neural cell to be treated and the neurological condition or disease.

The neural cells may be administered in a manner that allows them to be implanted into the desired tissue site and to reconstruct or regenerate the functionally defective region. For example, depending on the disease to be treated, the nerve cells may be transplanted directly into a parenchymal or intrathecal site into the central nervous system. In some embodiments, any of the neural cells described herein, including brain endothelial cells, neurons, dopamine neurons, ependymal cells, astrocytes, microglial cells, oligodendrocytes, and schwann cells are injected into a patient intravenously, intraventricularly, intrathecally, intraarterially, intramuscularly, intraperitoneally, subcutaneously, intramuscularly, intraperitoneally, intraocularly, postglobally, and combinations thereof. In some embodiments, the cells are injected or deposited in the form of a bolus or continuous infusion. In certain embodiments, the neural cells are administered by injection into the brain, the appropriate brain, and combinations thereof. The injection may be performed, for example, by drilling a hole in the skull of the subject. Suitable sites for administration of the neural cells to the brain include, but are not limited to, ventricles, lateral ventricles, large pool, putamen, basal nucleus, hippocampal cortex, striatum, caudate region of the brain, and combinations thereof.

Additional description of neural cells including dopamine neurons for use in the present technology can be found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

3. Endothelial cells differentiated from low immunity pluripotent cells

Provided herein are low immunity pluripotent cells that differentiate into various endothelial cell types for subsequent transplantation or implantation into a subject (e.g., recipient). As will be appreciated by those of ordinary skill in the art to which the invention pertains, the method of differentiation depends on the desired cell type using known techniques.

In some embodiments, endothelial cells differentiated from the subject low immunity pluripotent cells are administered to a patient in need thereof, e.g., a human patient. Endothelial cells may be administered to a patient suffering from a disorder or disease such as, but not limited to, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular occlusive disease, stroke, reperfusion injury, limb ischemia, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, vascular injury, tissue damage, hypertension, angina and myocardial infarction due to coronary artery disease, renal vascular hypertension, renal failure due to renal arterial stenosis, lower limb claudication, and the like. In certain embodiments, the patient has suffered from or is suffering from transient cerebral ischemia or stroke, which in some examples may be due to cerebrovascular disease. In some embodiments, the engineered endothelial cells are administered to treat tissue ischemia, e.g., as occurs in atherosclerosis, myocardial infarction, and limb ischemia, and repair damaged blood vessels. In some examples, the cells are used for bioengineering of the implant.

For example, endothelial cells can be used in cell therapies to repair ischemic tissue, form vascular and heart valves, engineering of vascular prostheses, repair damaged blood vessels, and induce the formation of blood vessels in engineered tissue (e.g., prior to implantation). In addition, endothelial cells may be further modified to deliver agents to the target and treat tumors.

In many embodiments, provided herein are methods of repairing or replacing tissue in need of vascular cells or angiogenesis. The method involves administering to a human patient in need of such treatment a composition comprising isolated endothelial cells to promote vascularization in the tissue. The tissue requiring vascular cells or vascularization may be heart tissue, liver tissue, pancreas tissue, kidney tissue, muscle tissue, nerve tissue, bone tissue, etc., which may be tissue that is damaged by cells and is characterized by excessive cell death, the risk of the tissue being damaged, or artificial engineering.

In some embodiments, vascular disease, possibly associated with heart disease or disorder, may be treated by administering endothelial cells, such as, but not limited to, final vascular endothelial cells and derived endocardial endothelial cells as described herein. Such vascular diseases include, but are not limited to, coronary artery disease, cerebrovascular disease, aortic stenosis, aortic aneurysm, peripheral arterial disease, atherosclerosis, varicose veins, vascular lesions, infarcted areas of the heart lacking coronary perfusion, non-healing wounds, diabetic or non-diabetic ulcers or any other disease or disorder requiring induction of angiogenesis.

In certain embodiments, endothelial cells are used to improve artificial implants (e.g., vessels made of synthetic materials, such as Dacron and gortex.) for use in vascular reconstructive surgery. For example, artificial arterial grafts are often used to replace diseased arteries perfusing vital organs or limbs. In other embodiments, engineered endothelial cells are used to cover the surface of the prosthetic heart valve to reduce the risk of embolization by making the valve surface less prone to thrombosis.

The endothelial cells outlined can be transplanted into the patient to transplant tissue and/or isolated cells into blood vessels using well known surgical techniques. In some embodiments, the cells are introduced into the heart tissue of the patient by injection (e.g., intramyocardial injection, intracoronary injection, transapical injection, percutaneous injection), infusion, transplantation, and implantation.

Administration (delivery) of endothelial cells includes, but is not limited to, subcutaneous or parenteral, including intravenous, intra-arterial (e.g., intra-coronary), intramuscular, intraperitoneal, intramyocardial, endocardial, epicardial, intranasal administration, and intrathecal, and infusion techniques.

As will be appreciated by those of ordinary skill in the art to which the invention pertains, HIP derivatives are transplanted using techniques known in the art to which the invention pertains, depending on the cell type and the end use of these cells. In some embodiments, the cells are transplanted at a particular location in the patient by intravenous or injection. When transplanted in a specific location, the cells may be suspended in a gel matrix to prevent them from dispersing upon fixation.

Exemplary endothelial cell types include, but are not limited to, microvascular endothelial cells, vascular endothelial cells, aortic endothelial cells, arterial endothelial cells, venous endothelial cells, renal endothelial cells, brain endothelial cells, liver endothelial cells, and the like.

The endothelial cells outlined herein may express one or more endothelial cell markers. Non-limiting examples of such markers include VE-adhesion protein (CD 144), ACE (angiotensin-converting enzyme) (CD 143), BNH9/BNF13, CD31, CD34, CD54 (ICAM-l), CD62E (E-selectin), CD105 (endothelial factor (Endoglin)), CD146, endothelial cell specific molecule (Endocan) (ESM-1), endoglyx-1, endostatin (Endomucin), eosin-3, EPAS1 (endothelial PAS domain protein 1), factor VIII-related antigen, FLI-l, flk-l (KDR, VEGFR-2), FLT-l (VEGFR-l), GATA2, GBP-l (guanylate-binding protein-l), GRO-alpha, HEX, ICAM-2 (intercellular adhesion molecule 2), LM02, LY-l, MRB (magic circle), nucleolin, PAL-E (pathologische anatomie Leiden-rtKTM), sVCM-1 (TEM-l, TALI, TEM), tumor markers (TEM-5), tumor markers (TEM-5), CD 141), VCAM-1 (vascular cell adhesion molecule-1) (CD 106), VEGF, vWF (Feng Wei ril (von Willebrand) factor), ZO-l, endothelial cell-selective adhesion molecule (ESAM), CD102, CD93, CD184, CD304 and DLL4.

In some embodiments, the endothelial cells are genetically modified to express exogenous genes encoding a protein of interest, such as, but not limited to, enzymes, hormones, receptors, ligands, or drugs useful in treating or ameliorating symptoms of a disorder/condition. Standard methods for genetically modifying endothelial cells are described, for example, in US 5,674,722.

Such endothelial cells can be used to provide constitutive synthesis and delivery of polypeptides or proteins useful for preventing or treating diseases. As such, the polypeptide is secreted directly into the subject's blood stream or other body area (e.g., central nervous system). In some embodiments, endothelial cells may be modified to secrete insulin, hemagglutinin (e.g., factor VIII or Feng Wei Rich (von Willebrand) factor), alpha-l antitrypsin, adenylate deaminase, tissue cytoplasmic pro-activator, metaalbumin (e.g., IL-1, IL-2, IL-3), and the like.

In some embodiments, endothelial cells may be modified in a manner that improves their performance in the context of implantation of the graft. Non-limiting illustrative examples include secretion or expression of thrombolytic agents to prevent blood clot formation in the lumen of blood vessels, secretion of inhibitors of smooth muscle proliferation to prevent luminal narrowing due to smooth muscle hypertrophy and expression and/or secretion of endothelial cell mitogens or autocrine factors to stimulate endothelial cell proliferation and improve the extent or duration of endothelial cell lining of the lumen of the graft.

In some embodiments, the engineered inner cell skin is used to deliver therapeutic amounts of secreted products to a specific organ or limb. For example, vascular implants lined with in vitro engineered (transduced) endothelial cells can be transplanted into a particular organ or limb. The secreted product of the transduced endothelial cells will be delivered to the perfused tissue in high concentrations to achieve the desired effect at the targeted anatomical site.

In other embodiments, the endothelial cells are genetically modified to include genes that interrupt or inhibit angiogenesis when expressed by the endothelial cells in the vascularized tumor. In some examples, endothelial cells may also be genetically modified to express any of the selective suicide genes described herein, which allow for negative selection of implanted endothelial cells after tumor treatment is complete.

In some embodiments, endothelial cells described herein are administered to the recipient subject to treat a vascular disorder selected from the group consisting of: vascular injury, cardiovascular disease, vascular disease, peripheral vascular disease, ischemic disease, myocardial infarction, congestive heart failure, peripheral vascular occlusion disease, hypertension, ischemic tissue injury, reperfusion injury, limb ischemia, stroke, neuropathy (e.g., peripheral neuropathy or diabetic neuropathy), organ failure (e.g., liver failure, kidney failure, etc.), diabetes, rheumatoid arthritis, osteoporosis, cerebrovascular disease, hypertension, angina and myocardial infarction due to coronary artery disease, renal vascular hypertension, renal failure due to renal arterial stenosis, lower limb lameness, and/or other vascular conditions or diseases.

In some embodiments, the low immunity pluripotent cells differentiate into Endothelial Cell Foci (ECFC) to form new blood vessels to address peripheral arterial disease. Techniques for differentiating endothelial cells are known. See, for example, prasain et al, doi:10.1038/nbt.3048, incorporated herein by reference in its entirety, and in particular methods and reagents for the production of endothelial cells from human pluripotent stem cells, and transplantation techniques. Differentiation can be assayed as known in the art, generally by assessing the presence of endothelial cell-associated or specific markers, or by measuring functionality.

In some embodiments, a method of producing a population of low-immunity endothelial cells from a population of low-immunity pluripotent cells by in vitro differentiation comprises (a) culturing a population of HIP cells in a first medium comprising a GSK inhibitor; (b) Culturing a population of HIP cells in a second medium comprising VEGF and bFGF to produce a population of pre-endothelial cells; and (c) culturing the population of pre-endothelial cells in a third medium comprising a ROCK inhibitor and an ALK inhibitor to produce a population of low-immunity endothelial cells.

In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some examples, the concentration of GSK inhibitor ranges from about 1mM to about 10mM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some examples, the concentration of ROCK inhibitor ranges from about 1pM to about 20pM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some examples, the concentration of ALK inhibitor ranges from about 0.5pM to about 10pM.

In some embodiments, the first medium comprises CHIR-99021 from 2pM to about 10 pM. In some embodiments, the second medium comprises 50ng/ml VEGF and 10ng/ml bFGF. In other embodiments, the second medium further comprises Y-27632 and SB-431542. In various embodiments, the third medium comprises 10pM Y-27632 and 1pM SB-431542. In certain embodiments, the third medium further comprises VEGF and bFGF. In certain examples, the first medium and/or the second medium is insulin-free.

The cells provided herein can be cultured on a surface, such as a synthetic surface that supports and/or promotes differentiation of low immunity pluripotent cells into cardiac cells. In some embodiments, the surface comprises a polymeric material including, but not limited to, homopolymers or copolymers of selected one or more acrylate monomers. Non-limiting examples of acrylate monomers and methacrylate monomers include tetra (ethylene glycol) diacrylate, glycerol dimethacrylate, 1, 4-butanediol dimethacrylate, poly (ethylene glycol) diacrylateDi (ethylene glycol) dimethacrylate, tetra (ethylene glycol) dimethacrylate, 1, 6-hexanediol propoxylate diacrylate, neopentyl glycol diacrylate, trimethylolpropane benzoate diacrylate, trimethylolpropane ethoxylate (1 EO/QH) methyl ester, tricyclo [5.2.1.0 ^2,6 ]Decanedimethanol diacrylate, neopentyl glycol ethoxylate diacrylate and trimethylolpropane triacrylate. Acrylates are synthesized as known in the art of the invention or are obtained from commercial suppliers such as Polysciences, inc, sigma Aldrich, inc.

In some embodiments, endothelial cells may be seeded onto the polymer matrix. In some examples, the polymer matrix is biodegradable. Suitable biodegradable matrices are well known in the art to which the invention pertains and include collagen-GAGs, collagen, fibrin, PLA, PGA and PLA/PGA copolymers. Additional biodegradable materials include poly (anhydride), poly (hydroxy acid), poly (orthoester), poly (propyl fumarate), poly (caprolactone), polyamides, polyamino acids, polyacetals, biodegradable polycyanoacrylates, biodegradable polyurethanes, and polysaccharides.

Non-biodegradable polymers may also be used. Other non-biodegradable but biocompatible polymers include polypyrrole, polyaniline, polythiophene, polystyrene, polyester, non-biodegradable polyurethane, polyurea, poly (ethylene vinyl acetate), polypropylene, polymethacrylate, polyethylene, polycarbonate, and poly (ethylene oxide). The polymer matrix may be formed in any shape, for example, as particles, sponges, tubes, spheres, wires, coiled wires, capillary networks, films, fibers, screens, or sheets. The polymer matrix may be modified to include natural or synthetic extracellular matrix materials and factors.

In some examples, the plastic or polymer includes dendritic polymers including poly (vinyl chloride), poly (vinyl alcohol), poly (methyl methacrylate), poly (vinyl acetate-maleic anhydride), poly (dimethylsiloxane), monomethacrylate, cyclic olefin polymers, fluorocarbon polymers, polystyrene, polypropylene, polyethyleneimine or derivatives of these, and the like. In some examples, the copolymer includes poly (vinyl acetate-co-maleic anhydride), poly (styrene-co-maleic anhydride), poly (ethylene-co-acrylic acid), derivatives of these, or the like.

In some embodiments, a population of low immunity endothelial cells is isolated from non-endothelial cells. In some embodiments, an isolated population of low-immunity endothelial cells is expanded prior to administration. In certain embodiments, prior to administration, an isolated population of low-immunity endothelial cells is expanded and cryopreserved.

Additional description of endothelial cells for use in the methods provided herein is found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

4. Thyroid cells differentiated from low immunity pluripotent cells

In some embodiments, the low immunity pluripotent cells differentiate into thyroid progenitor cells and thyroid follicular organelles that secrete thyroid hormones to address autoimmune thyroiditis. Techniques for differentiating thyroid cells are known in the art to which the invention pertains. See, e.g., kumann et al, cell Stem Cell,2015nov 5;17 527-42, methods and reagents for generating thyroid cells, particularly from human pluripotent stem cells, and transplantation techniques. Differentiation can be assayed as known in the art, typically by assessing the presence of thyroid cell-associated or specific markers or by measuring functionality.

5. Hepatocytes differentiated from low immunity pluripotent cells

In some specific embodiments, the low immunity pluripotent cells differentiate into hepatocytes to address loss of hepatocyte function or cirrhosis of the liver. There are a number of techniques available for differentiating HIP cells into hepatocytes; see, for example, pettinato et al, doi:10.1038/spre32888, snykers et al Methods Mol Biol 2011 698:305-314, si-Tayeb et al Hepatology 2010,51:297-305 and Asgari et al, stem Cell Rev,2013,9 (4): 493-504, which are incorporated herein by reference in their entirety, and in particular methods and reagents for differentiation. Differentiation may be assayed as known in the art, typically by assessing the presence of hepatocyte-related and/or specific markers, including, but not limited to, albumin, alpha fetoprotein, and fibrinogen. Differentiation can also be measured functionally, such as ammonia metabolism, LDL storage and uptake, ICG uptake and release, and hepatic glucose storage.

6. Pancreatic islet cells differentiated from low immunity pluripotent cells

In some embodiments, the pancreatic islet cells (also referred to as pancreatic beta cells) are derived from HIP cells described herein. In some examples, the low immunity pluripotent cells differentiated into various pancreatic islet cell types are transplanted or implanted into a subject (e.g., recipient). As will be appreciated by those of ordinary skill in the art to which the invention pertains, the method of differentiation depends on the desired cell type using known techniques. Exemplary pancreatic islet cell types include, but are not limited to, pancreatic islet progenitor cells, immature pancreatic islet cells, mature pancreatic islet cells, and the like. In some embodiments, pancreatic cells described herein are administered to a subject to treat diabetes.

In some embodiments, the pancreatic islet cells are derived from low immunity pluripotent cells described herein. Useful methods of differentiating pluripotent stem cells into pancreatic islet cells are described, for example, in US 9,683,215; US 9,157,062; and US 8,927,280.

In some embodiments, the pancreatic islet cells produced by the methods disclosed herein secrete insulin. In some embodiments, the pancreatic islet cells exhibit at least two characteristics of endogenous pancreatic islet cells, such as, but not limited to, secretion of insulin in response to glucose and expression of beta cell markers.

Exemplary beta cell markers or beta cell precursor markers include, but are not limited to, c-peptide, pdxl, glucose transporter 2 (Glut 2), HNF6, VEGF, glucokinase (GCK), prohormone converting enzyme (PC 1/3), cdcpl, neuroD, ngn3, nkx2.2, nkx6.1, nkx6.2, pax4, pax6, ptfla, isll, sox9, soxl7, and FoxA2.

In some embodiments, the isolated pancreatic islet cells produce insulin in response to increased glucose. In various embodiments, isolated pancreatic islet cells analyze insulin in response to increased glucose. In some embodiments, the cells have a distinct morphology, such as a pebble cell morphology and/or a diameter of about 17pm to about 25 pm.

In some embodiments, the low immunity pluripotent cells differentiate into beta-like cells or islets for transplantation to address type I diabetes (T1 DM). Cell systems are a promising approach to solving T1DM, see, for example, ellis et al, nat Rev Gastroenterol hepatol.2017, month 10; 14 (10): 612-628, incorporated herein by reference. Furthermore, pagliuca et al, (Cell, 2014,159 (2): 428-39), the contents of which are incorporated herein by reference in their entirety, report successful differentiation of beta-cells from hiPSCs, and in particular the methods and reagents for large-scale production of functional human beta cells from human pluripotent stem cells are summarized. Furthermore, vegas et al, show that human pluripotent stem cells produce human beta cells, which are then packaged to avoid immune rejection by the host; vegas et al, nat Med,2016,22 (3): 306-11, which are incorporated herein by reference in their entirety, and in particular the description outlines methods and reagents for large-scale production of functional human beta cells from human pluripotent stem cells.

In some embodiments, a method of generating a population of low-immunity pancreatic islet cells from a population of low-immunity pluripotent cells by in vitro differentiation comprises: (a) Culturing a population of HIP cells in a first medium comprising one or more factors selected from the group consisting of: insulin-like growth factors, transforming growth factors, FGF, EGF, HGF, SHH, VEGF, transforming growth factor-b superfamily, BMP2, BMP7, GSK inhibitor, ALK inhibitor, BMP type 1 receptor inhibitor, and retinoic acid to produce a population of immature pancreatic islet cells; and (b) culturing the population of immature pancreatic islet cells in a second medium different from the first medium to produce a population of low-immune pancreatic islet cells. In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof or variant thereof. In some examples, the concentration of GSK inhibitor ranges from about 2mM to about 10mM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some examples, the concentration of ALK inhibitor ranges from about 1pM to about 10pM. In some embodiments, neither the first medium nor the second medium is animal serum free.

In some embodiments, a population of low immunity pancreatic islet cells is isolated from non-pancreatic islet cells. In some embodiments, prior to administration, an isolated population of low-immunity pancreatic islet cells is expanded. In certain embodiments, prior to administration, an isolated population of low-immunity pancreatic islet cells is expanded and frozen.

Differentiation can be assayed as known in the art, generally by assessing the presence of a marker associated with or specific for beta cells, including but not limited to insulin. Differentiation may also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al, cell Syst.2016Oct 26;3 (4): 385-394.E3, which is incorporated by reference herein in its entirety, and in particular the biomarkers outlined herein. Once the beta cells are produced, they can be transplanted (in cell suspension or in a gel matrix as discussed herein) into the portal vein/liver, omentum, gastrointestinal mucosa, bone marrow, muscle, or subcutaneous sac.

Additional description of pancreatic islet cells including dopamine neurons for use in the present technology is found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

7. Retinal Pigment Epithelial (RPE) cells differentiated from low immunity pluripotent cells

Provided herein are Retinal Pigment Epithelial (RPE) cells derived from the HIP cells described above. For example, human RPE cells can be produced by differentiating human HIP cells. In some embodiments, the low immunity pluripotent cells differentiated into various RPE cell types are transplanted or implanted into a subject (e.g., recipient). As will be appreciated by those of ordinary skill in the art to which the invention pertains, the method of differentiation depends on the desired cell type using known techniques.

The term "RPE" cells refers to pigment retinal epithelial cells having a similar or substantially similar gene expression profile as the native RPE cells. This RPE derived from pluripotent stem cells may have a polygonal, planar lamellar morphology of natural RPE cells when grown to confluence on a planar substrate.

RPE cells may be implanted into a patient suffering from macular degeneration or a patient with damaged RPE cells. In some embodiments, the patient has age-related macular degeneration (AMD), early AMD, intermediate AMD, advanced AMD, non-neovascular age-related macular degeneration, dry macular degeneration (dry age-related macular degeneration), wet macular degeneration (wet age-related macular degeneration), juvenile Macular Degeneration (JMD) (e.g., stargardt disease, best disease, and retinal tear), leber's congenital black barrier, or retinitis pigmentosa. In other embodiments, the patient has retinal detachment.

Exemplary RPE cell types include, but are not limited to, retinal Pigment Epithelial (RPE) cells, RPE progenitor cells, immature RPE cells, mature RPE cells, functional RPE cells, and the like.

Useful methods of differentiating pluripotent stem cells into RPE cells are described, for example, in US9,458,428 and US9,850,463, the disclosures of which are incorporated herein by reference in their entirety, including the description. Additional methods for generating RPE cells from human induced pluripotent stem cells can be found in, for example, lamba et al, PNAS,2006,103 (34): 12769-12774; mellough et al, stem Cells,2012,30 (4): 673-686; idelson et al, cell Stem Cell,2009,5 (4): 396-408; rowland et al Journal of Cellular Physiology,2012,227 (2): 457-466, buchholz et al Stem Cells Trans Med,2013,2 (5): 384-393 and da Cruz et al, nat Biotech,2018,36: 328-337.

Human pluripotent stem cells have been differentiated into RPE cells using the techniques outlined in Kamao et al Stem Cell Reports 2014:2:205-18, hereby incorporated by reference in its entirety, and methods and reagents for differentiation techniques and reagents are specifically outlined for the same; see also Mandai et al, N Engl J Med,2017,376:1038-1046, which are incorporated by reference in their entirety for the production of RPE cell sheets and transplantation into patients. Differentiation may be assayed as known in the art, typically by assessing the presence of RPE-related and/or specific markers or by measuring functionality. See, e.g., kamao et al, stem Cell Reports,2014,2 (2): 205-18, the contents of which are incorporated by reference in their entirety, and specifically for the labels outlined in the first paragraph of the results section.

In some embodiments, a method of producing a population of low immunity Retinal Pigment Epithelial (RPE) cells from a population of low immunity pluripotent cells by in vitro differentiation comprises: (a) Culturing a population of low immunity pluripotent cells in a first medium comprising any one of the factors selected from the group consisting of: activin A, bFGF, BMP/7, DKK1, IGF1, noggin, BMP inhibitor, ALK inhibitor, ROCK inhibitor, and VEGFR inhibitor to produce a population of pre-RPE cells; and (b) culturing the population of pre-RPE cells in a second medium different from the first medium to produce a population of low immunity RPE cells. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some examples, the concentration of ALK inhibitor ranges from about 2mM to about 10pM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some examples, the concentration of ROCK inhibitor ranges from about 1pM to about 10pM. In some embodiments, neither the first medium nor the second medium is animal serum free.

Differentiation can be assayed as known in the art, typically by assessing the presence of RPE-related or specific markers or by measuring functionality. See, e.g., kamao et al, stem Cell Reports,2014,2 (2): 205-18, the contents of which are incorporated herein by reference in their entirety, and in particular the results section.

An additional description of RPE cells useful in the present technology is found in WO2020/018615, the disclosure of which is incorporated herein by reference in its entirety.

For therapeutic applications, cells prepared according to the disclosed methods may typically be provided in the form of pharmaceutical compositions comprising isotonic excipients, and prepared under conditions sufficiently sterile for human administration. For general principles in pharmaceutical formulations of Cell compositions, see Morstyn & Sheridan eds, cambridge University Press,1996, "Cell Therapy: stem Cell Transplantation, gene Therapy, and Cellular Immunotherapy"; and E.D.Ball, J.Lister & p.law, churchill Livingstone,2000, "Hematopoietic Stem Cell Therapy". The cells may be packaged in a device or container suitable for dispensing or clinical use.

8. T lymphocytes derived from low immunity pluripotent cells

Provided herein are T lymphocytes (T cells, including primary T cells) that are derived from HIP cells (e.g., low immunity ipscs) described herein. Methods of generating T cells, including CAR-T-cells, from pluripotent stem cells (e.g., ipscs), are described, for example, in irigchi et al, nature Communications, 430 (2021); themeli et al, 16 (4): 357-366 (2015); themeli et al Nature Biotechnology 31:928-933 (2013). T lymphocyte-derived hypo-immune cells include, but are not limited to, primary T cells that escape immune recognition. In some embodiments, the low immunity cells are generated (e.g., produced, cultured, or derived) from T cells, such as primary T cells. In some examples, the primary T cells are obtained (e.g., obtained, extracted, removed, or retrieved) from a subject or individual. In some embodiments, the primary T cells are generated from a T cell pool such that the T cells are from one or more subjects (e.g., one or more humans, including one or more healthy humans). In some embodiments, the primary T cell pool is from 1 to 100, 1 to 50, 1 to 20, 1 to 10, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, or 100 or more subjects. In some embodiments, the donor subject is different from the patient (e.g., recipient of the administered therapeutic cells). In some embodiments, the T cell pool does not include cells from the patient. In some embodiments, one or more donor subjects from whom the T cell pool is obtained are different from the patient.

In some embodiments, the low-immunity cells do not activate the immune response of the patient (e.g., the recipient after administration). Methods are provided for treating a disorder by administering a population of low immunity cells to a subject (e.g., recipient) or patient in need thereof. In some embodiments, the low immunity cells described herein comprise T cells engineered (e.g., modified) to express a chimeric antigen receptor, including but not limited to the chimeric antigen receptor described herein. In some examples, the T cells are from a population or subpopulation of primary T cells from one or more individuals. In some embodiments, a T cell described herein, such as an engineered or modified T cell, comprises an endogenous T cell receptor that reduces expression.

In some embodiments, the HIP-derived T cells comprise a Chimeric Antigen Receptor (CAR). Any suitable CAR may be included in a HIP-derived T cell, including a CAR as described herein. In some embodiments, the HIP-derived T cell comprises a polynucleotide encoding a CAR, wherein the polynucleotide is inserted at a genomic locus. In some embodiments, the polynucleotide is inserted into a safe harbor or target locus. In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene. Any suitable method can be used to insert the CAR into the genomic locus of the low immunity cell, including the gene editing methods described herein (e.g., CRISPR/Cas system).

HIP-derived T cells provided herein are useful for treating suitable cancers, including, but not limited to, B-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

9. NK cells derived from low immunity pluripotent cells

Provided herein are Natural Killer (NK) cells derived from HIP cells (e.g., low-immunity ipscs) described herein.

NK cells (also defined as "large granular lymphocytes") represent cell lineages that differentiate from common lymphocyte precursors (which also produce B lymphocytes and T lymphocytes). Unlike T cells, NK cells do not naturally contain CD3 on the cell membrane. Importantly, NK cells do not express TCRs and typically also lack other antigen-specific cell surface receptors (as well as TCRs and CD3, which do not express immunoglobulin B cell receptors, but typically express CD16 and CD 56). NK cell cytotoxic activity does not require sensitization but is enhanced by activation of various cytokines including IL-2. NK cells are generally thought to lack the appropriate or complete signaling pathway required for antigen receptor-mediated signaling and are therefore not thought to be capable of antigen receptor-dependent signaling, activation and expansion. NK cells are cytotoxic and balance activation and inhibition of receptor signaling to modulate their cytotoxic activity. For example, NK cells expressing CD16 may bind to the Fc domain of an antibody that the infected cells bind to, resulting in NK cell activation. In contrast, cellular activity is reduced for the expression of high amounts of MHC class I proteins. In contact with the target cells, NK cells release proteins, such as perforins and enzymes, such as proteases (granzymes). Perforin can form a hole in the cell membrane of the target cell, induce apoptosis or cytolysis.

There are many techniques available for producing NK cells, including CAR-NK-cells, from pluripotent stem cells (e.g., ipscs); see, e.g., zhu et al, methods Mol biol.2019;2048:107-119; knorr et al Stem Cells Transl Med.2013 2 (4): 274-83.Doi:10.5966/sctm.2012-0084; zeng et al, stem Cell reports.2017dec12;9 (6) 1796-1812; ni et al, methods Mol biol.2013;1029:33-41; bernareggi et al, exp Hematol.2019:13-23; shankar et al, stem Cell Res ter 2020;11 234, all of which are incorporated herein by reference in their entirety, and in particular methods and reagents for differentiation. Differentiation may be assayed as known in the art, generally by assessing the presence of NK cell-associated and/or specific markers, including, but not limited to, CD56, KIRs, CD16, NKp44, NKp46, NKG2D, TRAIL, CD122, CD27, CD244, NK1.1, NKG2A/C, NCR1, ly49, CD49b, CD11b, KLRG1, CD43, CD62L, and/or CD226.

In some embodiments, the low immunity pluripotent cells differentiate into hepatocytes to address hepatocyte loss of function or cirrhosis. There are a number of techniques available for differentiating HIP cells into hepatocytes; see, for example, pettinato et al, doi:10.1038/spre32888, snykers et al, methods Mol biol., 2011:698-305, si-Tayeb et al, hepatology,2010,51:297-305 and Asgari et al, stem Cell rev.,2013,9 (4): 493-504, all incorporated herein by reference in their entirety, and particularly for methods and reagents for differentiation. Differentiation may be assayed as known in the art, typically by assessing the presence of hepatocyte-related and/or specific markers, including, but not limited to, albumin, alpha fetoprotein, and fibrinogen. Differentiation may also be measured functionally, such as ammonia metabolism, LDL storage and uptake, ICG uptake and release, and hepatic glucose storage.

In some embodiments, NK cells do not activate the immune response of the patient (e.g., the recipient after administration). Methods are provided for treating a disorder by administering a population of NK cells to a subject (e.g., recipient) or patient in need thereof. In some embodiments, the NK cells described herein comprise NK cells engineered (e.g., modified) to express a chimeric antigen receptor, including but not limited to the chimeric antigen receptor described herein. Any suitable CAR may be included in NK cells, including the CARs described herein. In some embodiments, the NK cell comprises a polynucleotide encoding a CAR, wherein the polynucleotide is inserted at a genomic locus. In some embodiments, the polynucleotide is inserted into a safe harbor or target locus. In some embodiments, the polynucleotide is inserted into the B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene. Any suitable method can be used to insert the CAR into the genomic locus of the NK cell, including the gene editing methods described herein (e.g., CRISPR/Cas system).

U.exogenous polynucleotide

In some embodiments, the low immunity cells provided herein are genetically modified to include one or more exogenous polynucleotides inserted into one or more genomic loci of the low immunity cells. In some embodiments, the exogenous polynucleotide encodes a protein of interest, e.g., a chimeric antigen receptor. Any suitable method can be used to insert the exogenous polynucleotide into the genomic locus of the low immunity cell, including the gene editing methods described herein (e.g., CRISPR/Cas system).

The exogenous polynucleotide may be inserted into the genomic locus of any suitable low immunity cell. In some embodiments, the exogenous polynucleotide is inserted into a safe harbor or target locus as described herein. Suitable safe harbors and target loci include, but are not limited to, CCR5 genes, CXCR4 genes, PPP1R12C (also known as AAVS 1) genes, albumin genes, SHS231 loci, CLYBL genes, rosa genes (e.g., rosa 26), F3 genes (also known as CD 142), MICA genes, MICB genes, LRP1 genes (also known as CD 91), HMGB1 genes, ABO genes, RHD genes, FUT1 genes, PDGFRa genes, OLIG2 genes, GFAP genes, and KDM5D genes (also known as HY). In some embodiments, the exogenous polynucleotide is inserted into an intron, an exon, or a coding sequence region of a safe harbor or target gene locus. In some embodiments, the exogenous polynucleotide is inserted into an endogenous gene, wherein the insertion results in silencing or reduced expression of the endogenous gene. In some embodiments, the polynucleotide is inserted at the B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene locus. Exemplary genomic loci for exogenous polynucleotide insertions are described in tables 4 and 5.

TABLE 4 exemplary genomic loci for exogenous polynucleotide insertion

TABLE 5 non-limiting examples of Cas9 guide RNAs

For Cas9 guidance, all Cas 9-guided spacer sequences are provided in table 6, with the 20nt guide sequence described corresponding to a unique guide sequence and may be any of those described herein, including, for example, those listed in table 6.

TABLE 6 Cas9 guide RNA

In some embodiments, the low immunity cells comprising the exogenous polynucleotide are derived from low immunity pluripotent cells (HIPs), e.g., as described herein. Such low immunity cells include, for example, cardiac cells, neural cells, brain endothelial cells, dopamine neurons, glial cells, endothelial cells, thyroid cells, pancreatic islet cells (beta cells), retinal pigment epithelial cells, and T cells. In some embodiments, the low immunity cell comprising the exogenous polynucleotide is a pancreatic β cell, a T cell (e.g., a primary T cell), or a glial progenitor cell.

In some embodiments, the exogenous polynucleotide encodes an exogenous CD47 polypeptide (e.g., a human CD47 polypeptide) and the exogenous polypeptide is inserted into a safe harbor or target locus or a safe harbor or target locus disclosed herein or a genomic locus of an endogenous gene that results in silencing or reduced expression. In some embodiments, the polynucleotide is inserted at the B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene locus. In some embodiments, the gene encoding CD47 is inserted into a specific locus selected from the group consisting of: a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, a TRB locus, a PD1 locus, and a CTLA4 locus. In some embodiments, the gene encoding the CAR is inserted into a specific locus selected from the group consisting of: a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the gene encoding CD47 and the gene encoding CAR are inserted into different loci. In some embodiments, the gene encoding CD47 and the gene encoding CAR are inserted into the same locus. In some embodiments, the gene encoding CD47 and the gene encoding CAR are inserted into the B2M locus, CIITA locus, TRAC locus, TRB locus, or safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of: CCR5 gene locus, CXCR4 gene locus, PPP1R12C gene locus, albumin gene locus, SHS231 gene locus, CLYBL gene locus, rosa gene locus, F3 (CD 142) gene locus, MICA gene, locus MICB gene, locus LRP1 (CD 91) gene locus, HMGB1 gene locus, ABO gene locus, RHD gene locus, FUT1 gene locus, PDGFRa gene locus, OLIG2 gene locus, GFAP gene locus and KDM5D gene locus.

In some embodiments, the low immunity cells comprising the exogenous polynucleotide are primary T cells or T cells derived from low immunity pluripotent cells (e.g., low immunity ipscs). In exemplary embodiments, the exogenous polynucleotide is a chimeric antigen receptor (e.g., any of the CARs described herein). In some embodiments, the exogenous polynucleotide is operably linked to a promoter to express the exogenous polynucleotide in the low immunity cell.

In some embodiments, the low immunity cell comprising the exogenous polynucleotide is a primary T cell or a T cell derived from a low immunity pluripotent cell (e.g., a low immunity iPSC) and comprises a first exogenous polynucleotide encoding a CAR polypeptide and a second exogenous polynucleotide encoding a CD47 polypeptide. In some embodiments, the first exogenous polynucleotide is inserted into the same genomic locus with a second exogenous polynucleotide. In some embodiments, the first exogenous polynucleotide is inserted into a different genomic locus with a second exogenous polynucleotide. In exemplary embodiments, the low immunity cells are primary T cells or T cells derived from low immunity pluripotent cells (e.g., ipscs).

In some embodiments, the low immunity cells comprising the exogenous polynucleotide are primary NK cells or NK cells derived from low immunity pluripotent cells (e.g., low immunity ipscs). In exemplary embodiments, the exogenous polynucleotide is a chimeric antigen receptor (e.g., any of the CARs described herein). In some embodiments, the exogenous polynucleotide is operably linked to a promoter to express the exogenous polynucleotide in the low immunity cell. In some embodiments, the low immunity cell comprising the exogenous polynucleotide is a primary NK cell or NK cell derived from a low immunity pluripotent cell (e.g., a low immunity iPSC) and comprises a first exogenous polynucleotide encoding a CAR polypeptide and a second exogenous polynucleotide encoding a CD47 polypeptide. In some embodiments, the first exogenous polynucleotide is inserted into the same genomic locus with a second exogenous polynucleotide. In some embodiments, the first exogenous polynucleotide is inserted into a different genomic locus with a second exogenous polynucleotide. In exemplary embodiments, the low immunity cells are primary NK cells or NK cells derived from low immunity pluripotent cells (e.g., ipscs).

In some embodiments, the low immunity cell comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different exogenous polynucleotides inserted into one or more genomic loci as described herein (e.g., table 4). In some embodiments, the exogenous polynucleotide is inserted into the same genomic locus. In some embodiments, the exogenous polynucleotide is inserted into a different genomic locus.

In some embodiments, the exogenous polynucleotide encodes one of the following factors: DUX4, CD24, CD27, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, fasL, CCL21, CCL22, mfge8, serpin 9, and any tolerogenic factors provided herein.

V. transplantation of cells

As will be appreciated by those of ordinary skill in the art to which the invention pertains, cells and derivatives thereof may be transplanted using techniques known in the art to which the invention pertains, depending on the cell type and end use of the cells. In general, the cells described herein may be transplanted at a particular location in a patient, either intravenously or by injection. When transplanted at a particular location, cells may be suspended in a gel matrix to prevent their dispersion upon fixation.

W immunosuppressant

In some embodiments, the immunosuppressive and/or immunomodulatory agent is not administered to the patient prior to the first administration of the population of low-immunity cells. In many embodiments, the immunosuppressive and/or immunomodulatory agent is administered to the patient prior to the first administration of the population of low-immunity cells. In some embodiments, the immunosuppressive and/or immunomodulatory agent is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more prior to the first administration of the cells. In some embodiments, the immunosuppressive and/or immunomodulatory agent is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more prior to the first administration of the cells. In certain embodiments, the immunosuppressive and/or immunomodulatory agent is not administered to the patient after the first administration of the cells or is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more after the first administration of the cells. In some embodiments, the immunosuppressive and/or immunomodulatory agent is administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more after the first administration of the cells. Non-limiting examples of immunosuppressive and/or immunomodulatory agents include cyclosporine, azathioprine, mycophenolic acid ester (mycophenolate mofetil), corticosteroids such as prednisone, methotrexate, clo Jin Huana (gold salt), sulfasalazine (sulfasalazine), antimalarial agents, butraline (brequar), leflunomide (leflunomide), mizoribine (mizoribine), 15-deoxyspergualin (deoxyspergualin), 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin (thymopentin), thymosin-alpha and the like. In some embodiments, the immunosuppressive and/or immunomodulatory agent is selected from the group of immunosuppressive antibodies consisting of: antibodies that bind to p75 of the IL-2 receptor, antibodies that bind to, for example, MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFN-gamma, TNF-. Alpha., IL-4, IL-5, IL-6R, IL-6, IGF, IGFR1, IL-7, IL-8, IL-10, CD11a or CD58, and antibodies that bind to any of their ligands. In some embodiments, when the immunosuppressive and/or immunomodulatory agent is administered to the patient before or after the first administration of the cells, then the administration is at a lower dose than is required for cells with MHC I and/or MHC II expression without expression of exogenous CD 47.

In one embodiment, the immunosuppressive and/or immunomodulatory agent can be selected from soluble IL-15R, IL-10, B7 molecules (e.g., B7-1, B7-2, variants and fragments thereof), ICOS and OX40, inhibitors of negative T cell modulators (such as antibodies to CTLA-4), and the like.

In some embodiments, the immunosuppressive and/or immunomodulatory agent is not administered to the patient prior to administration of the population of low-immunity cells. In many embodiments, the immunosuppressive and/or immunomodulatory agent is administered to the patient prior to the first and/or second administration of the population of low-immunity cells. In some embodiments, the immunosuppressive and/or immunomodulatory agent is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more prior to administration of the cells. In some embodiments, the immunosuppressive and/or immunomodulatory agent is administered for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more prior to the first and/or second administration of the cells. In certain embodiments, the immunosuppressive and/or immunomodulatory agent is administered for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more after administration of the cells. In some embodiments, the immunosuppressive and/or immunomodulatory agent is administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or longer after the first and/or second administration of the cells. In some embodiments, when the immunosuppressive and/or immunomodulatory agent is administered to the patient before or after administration of the cells, then administration is at a lower dose than is required for cells with MHC I and/or MHC II expression without expression of exogenous CD 47.

Detailed description of the preferred embodiments

In one aspect, provided herein is a method comprising administering to a patient a population of low immunity cells comprising an exogenous CD47 polypeptide and a reduced expression of MHC class I and/or II human leukocyte antigen, wherein the patient is sensitive to one or more alloantigens. In some embodiments, the method is for treating a disorder in a patient.

In some embodiments, the patient is susceptible to prior pregnancy or prior allograft. In some embodiments, the one or more alloantigens comprise human leukocyte antigens. In some embodiments, the patient exhibits a memory B cell and/or memory T cell response to one or more alloantigens. In some embodiments, the allograft is selected from the group consisting of: allogeneic cell grafts, allogeneic blood transfusion, allogeneic tissue grafts, and allogeneic organ grafts. In some embodiments, the patient exhibits reduced or no immune response to a population of low immunity cells. In some examples, the patient exhibits an immune response to the allograft and a reduced or no immune response to a population of low immune cells. In some embodiments, the reduced or no immune response is selected from the group consisting of: reduced or no systemic immune response, reduced or no adaptive immune response, reduced or no innate immune response, reduced or no T cell response, and reduced or no B cell response to a population of low immune cells.

In some embodiments, a population of low-immunity cells is administered for at least one week or more after the patient is sensitive to one or more alloantigens. In certain embodiments, a population of low-immunity cells is administered for at least 1 month or more after the patient is sensitive to one or more alloantigens.

In some embodiments, the low immunity cells comprise MHC class I and class II human leukocyte antigens that reduce expression. In some embodiments, the low immunity cell comprises an exogenous CD47 polypeptide and a reduced expression amount of B2M and/or CIITA. In some embodiments, the low immunity cell comprises an exogenous CD47 polypeptide and reduced expression levels of B2M and CIITA. In some embodiments, the low immunity cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and/or combinations thereof. In some embodiments, the low immunity cell further comprises a reduced amount of CD142.

In some embodiments, the low immunity cell is a differentiated cell derived from a pluripotent stem cell. In some embodiments, the pluripotent stem cells comprise induced pluripotent stem cells. In some embodiments, the differentiated cells are selected from the group consisting of: cardiac cells, neural cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells (e.g., plasma cells or platelets), and epithelial cells.

In some embodiments, the low immunity cells comprise cells derived from primary T cells. In some embodiments, the cells derived from the primary T cells are derived from a T cell pool comprising primary T cells from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) subjects other than the patient.

In some embodiments, the cells derived from primary T cells comprise a chimeric antigen receptor. In some embodiments, the Chimeric Antigen Receptor (CAR) is selected from the group consisting of: (a) A first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) A second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) A third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene following successful signaling of the CAR.

In some embodiments of the CAR, the antigen binding domain is selected from the group consisting of: (a) The antigen binding domain targets the antigenic properties of the tumor cell; (b) An antigen binding domain that targets the antigenic properties of T cells; (c) The antigen binding domain targets the antigenic properties of autoimmune or inflammatory disorders; (d) An antigen binding domain that targets an antigenic feature of senescent cells; (e) An antigen binding domain that targets an antigenic feature of an infectious disease; and (f) an antigen binding domain that binds to a cell surface antigen of a cell.

In some embodiments, the antigen binding domain of the CAR is selected from the group consisting of: antibodies, antigen binding portions thereof, scFv and Fab. In some embodiments, the antigen binding domain binds to CD19 or BCMA.

In some embodiments, the transmembrane domain of the CAR comprises one selected from the group consisting of: tcra, tcrβ, tcrζ, cd3ζ, cd3γ, cd3δ, cd3ζ, CD4, CD5, cd8α, cd8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40L/CD154, CD45, CD64, CD80, CD86, OX40/CD134, 4-1BB/CD137, CD154, fcsry, VEGFR2, FAS, the transmembrane region of FGFR2B, and functional variants thereof.

In some embodiments, the signaling domain of the CAR comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises two different co-stimulatory domains. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation.

For fourth generation CARs, it comprises a domain that induces cytokine gene expression following successful signaling of the CAR, in some embodiments, the cytokine gene is due to an endogenous or exogenous cytokine gene to the low immunity cell. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-9, IL-12, IL18, TNF, IFN-gamma and functional fragments thereof.

In some embodiments of the fourth generation CAR, the domain that induces cytokine gene expression following successful signaling of the CAR comprises a transcription factor or functional domain or fragment thereof.

In some embodiments of cells derived from primary T cells, the CAR comprises a CD3 zeta (CD 3 zeta) domain or an immunoreceptor tyrosine based activation motif (ITAM) or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes. In certain embodiments, the CAR comprises (i) an anti-CD 19 scFv; (ii) A CD8 a hinge and a transmembrane domain or functional variant thereof; (iii) a 4-1BB co-stimulatory domain or a functional variant thereof; and (iv) a CD3 zeta signaling domain or a functional variant thereof.

In some embodiments, the cells derived from primary T cells comprise an endogenous T cell receptor that reduces expression. In particular embodiments, the cells derived from primary T cells comprise reduced expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA 4) and/or programmed cell death (PD 1). In certain embodiments, the cells derived from primary T cells comprise an increased expression of programmed cell death ligand 1 (PD-L1).

In some embodiments of the method, a population of low immune cells causes a reduced amount of immune activation or no immune activation in the patient after administration. In certain embodiments, a population of low immune cells causes a reduced amount of systemic TH1 activation or no systemic TH1 activation in the patient after administration. In some embodiments, a population of low immune cells causes immune activation of reduced amounts of Peripheral Blood Mononuclear Cells (PBMCs) or no PBMCs in the patient after administration. In certain embodiments, a population of low immune cells causes a reduced amount of donor-specific IgG antibodies or no donor-specific IgG antibodies to the low immune cells in the patient after administration. In some embodiments, a population of low immune cells causes reduced or no IgM and IgG antibody production in the patient after administration to the low immune cells. In other embodiments, a population of low immunity cells causes a reduced amount of cytotoxic T cell killing or non-cytotoxic T cell killing of the low immunity cells in the patient after administration. In certain embodiments, a population of low immunity cells does not trigger a systemic acute cellular immune response in the patient after administration.

In some embodiments, the patient is not administered the immunosuppressant for at least 3 days or more before or after administering the population of low immunity cells.

In another aspect, provided herein is a method comprising administering to a patient a dosing regimen comprising: (a) A first administration comprising a therapeutically effective amount of low immunity cells; (b) during recovery; and (c) a second administration comprising a therapeutically effective amount of low immunity cells; wherein the low immunity cells comprise an exogenous CD47 polypeptide and a reduced expression of MHC class I and/or II human leukocyte antigen, and wherein the patient is sensitive to one or more alloantigens. In some embodiments, the methods are useful for treating a disorder in a patient.

In some embodiments, the patient is susceptible to prior pregnancy or prior allograft. In some embodiments, the one or more alloantigens comprise human leukocyte antigens. In some embodiments, the patient exhibits a memory B cell and/or memory T cell response to one or more alloantigens. In some embodiments, the allograft is selected from the group consisting of: allogeneic cell grafts, allogeneic blood transfusion, allogeneic tissue grafts, and allogeneic organ grafts.

In some embodiments, the patient exhibits reduced or no immune response to a population of low immunity cells. In some examples, the reduced or no immune response is selected from the group consisting of: reduced or no systemic immune response, reduced or no adaptive immune response, reduced or no innate immune response, reduced or no T cell response, and reduced or no B cell response to a population of low immune cells.

In some embodiments, the first administration of the low-immunity cells occurs at least one week or more after the patient is sensitive to the one or more alloantigens. In some embodiments, the first administration of the low-immunity cells occurs at least 1 month or more after the patient is sensitive to the one or more alloantigens.

In some embodiments, the low immunity cell further comprises MHC class I and class II human leukocyte antigens that reduce expression. In some embodiments, the low immunity cell expresses an exogenous CD47 polypeptide and a reduced amount of B2M and/or CIITA. In some embodiments, the low immunity cells express exogenous CD47 polypeptides and reduced expression levels of B2M and CIITA. In some embodiments, the low immunity cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof. In some embodiments, the low immunity cell further comprises a reduced amount of CD142.

In some embodiments, the low immunity cell is a differentiated cell derived from a pluripotent stem cell. In certain embodiments, the pluripotent stem cells comprise induced pluripotent stem cells. In many embodiments, the differentiated cells are selected from the group consisting of: cardiac cells, neural cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells (e.g., plasma cells or platelets), and epithelial cells.

In some embodiments, the low immunity cells comprise cells derived from primary T cells. In certain embodiments, the cells derived from the primary T cells are derived from a T cell pool comprising primary T cells from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) subjects other than the patient. In some embodiments, the cells derived from primary T cells comprise a chimeric antigen receptor.

In some embodiments, the Chimeric Antigen Receptor (CAR) is selected from the group consisting of: (a) A first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) A second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) A third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene following successful signaling of the CAR.

In some embodiments, the antigen binding domain is selected from the group consisting of: (a) The antigen binding domain targets the antigenic properties of the tumor cell; (b) An antigen binding domain that targets an antigenic property of a T cell, (c) an antigen binding domain that targets an antigenic property of an autoimmune or inflammatory disorder; (d) An antigen binding domain that targets an antigenic feature of senescent cells; (e) An antigen binding domain that targets an antigenic feature of an infectious disease; and (f) an antigen binding domain that binds to a cell surface antigen of a cell. In some embodiments, the antigen binding domain is selected from the group consisting of: antibodies, antigen binding portions thereof, scFv and Fab. In certain embodiments, the antigen binding domain binds to CD19 or BCMA.

In some embodiments, the transmembrane domain comprises one selected from the group consisting of: tcra, tcrβ, tcrζ, cd3ζ, cd3γ, cd3δ, cd3ζ, CD4, CD5, cd8α, cd8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40L/CD154, CD45, CD64, CD80, CD86, OX40/CD134, 4-1BB/CD137, CD154, fcsry, VEGFR2, FAS, the transmembrane region of FGFR2B, and functional variants thereof.

In some embodiments, the signaling domain comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises two different co-stimulatory domains. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation.

In some embodiments of the fourth generation CAR, successful signaling of the CAR induces expression of the cytokine gene. In some embodiments, the cytokine gene is an endogenous or exogenous cytokine gene to the low immunity cell. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-9, IL-12, IL18, TNF, IFN-gamma and functional fragments thereof. In some embodiments of the fourth generation CAR, the domain that induces cytokine gene expression following successful signaling of the CAR comprises a transcription factor or functional domain or fragment thereof.

In some embodiments, the CAR comprises a CD3 zeta (cd3ζ) domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes. In some embodiments, the CAR comprises (i) an anti-CD 19 scFv; (ii) A CD8 a hinge and a transmembrane domain or functional variant thereof; (iii) a 4-1BB co-stimulatory domain or a functional variant thereof; and (iv) a CD3 zeta signaling domain or a functional variant thereof.

In some embodiments, the cells derived from primary T cells comprise an endogenous T cell receptor that reduces expression. In some embodiments, the cells derived from primary T cells comprise reduced expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA 4) and/or programmed cell death (PD 1). In some embodiments, the cells derived from primary T cells comprise an increased expression of programmed cell death ligand 1 (PD-L1).

In some embodiments, the recovery period comprises at least 1 month or more (e.g., at least 1 month, 2 months, 3 months, 4 months or more). In some embodiments, the recovery period comprises at least 2 months or more (e.g., at least 2 months, 3 months, 4 months or more).

In some embodiments, the second administration of the cells is initiated when the low immunity cells from the first administration are no longer detectable in the patient.

In some embodiments, the low-immune cells cause a reduced amount of or no immune activation in the patient after the first and/or second administration (e.g., after the first administration or the second administration or after both the first and second administration). In some embodiments, the low immune cells cause a reduced amount of systemic TH1 activation or no systemic TH1 activation in the patient after the first and/or second administration. In some embodiments, the low immune cells cause immune activation of reduced amounts of Peripheral Blood Mononuclear Cells (PBMCs) or no PBMCs in the patient after the first and/or second administration. In some embodiments, the low-immune cells cause a reduced amount of donor-specific IgG antibodies or no donor-specific IgG antibodies in the patient against the low-immune cells after the first and/or second administration. In some embodiments, the low-immune cells cause reduced or no IgM and IgG antibody production in the patient against the low-immune cells after the first and/or second administration. In some embodiments, the low-immune cells cause a reduced amount of cytotoxic T cell killing or non-cytotoxic T cell killing of the low-immune cells in the patient after the first and/or second administration.

In some embodiments, the patient is not administered the immunosuppressant for at least 3 days or more before or after the first administration of the low immunity cells. In some embodiments, the patient is not administered the immunosuppressant for at least 3 days or more before or after the second administration of the low immunity cells. In certain embodiments, the patient is not administered an immunosuppressant during recovery.

In some embodiments, the methods described herein further comprise administering the dosing regimen at least twice. In some examples, the dosing regimen is administered at least 2 times (e.g., at least 2, 3, 4, or more times) to a patient susceptible to one or more alloantigens.

Provided herein is the use of a population of low immunity cells comprising an exogenous CD47 polypeptide and a reduced expression of MHC class I and/or II human leukocyte antigens for treating a disorder in a patient, wherein the patient is sensitive to one or more alloantigens.

Provided herein is the use of a population of low immunity cells comprising an exogenous CD47 polypeptide and reduced expression of MHC class I and class II human leukocyte antigens for treating a disorder in a patient, wherein the patient is sensitive to one or more alloantigens.

Provided herein is the use of a population of low immunity cells comprising an exogenous CD47 polypeptide and reduced amounts of B2M and CIITA polypeptides for treating a disorder in a patient, wherein the patient is sensitive to one or more alloantigens.

Provided herein is the use of a population of low immunity cells comprising exogenous CD47 polypeptide, genomic modification of B2M gene, and genomic modification of CIITA gene for treating a disorder in a patient, wherein the patient is sensitive to one or more alloantigens.

In some embodiments of the use of the population of cells, the one or more alloantigens comprise human leukocyte antigens. In some embodiments, the patient exhibits a memory B cell and/or memory T cell response to one or more alloantigens.

In some embodiments of the use, the patient is susceptible to prior pregnancy or prior allograft. In some embodiments, the allograft is selected from the group consisting of: allogeneic cell grafts, allogeneic blood transfusion, allogeneic tissue grafts, and allogeneic organ grafts.

In some embodiments, the patient exhibits reduced or no immune response to a population of low immunity cells. In certain embodiments, the reduced or no immune response is selected from the group consisting of: reduced or no systemic immune response, reduced or no adaptive immune response, reduced or no innate immune response, reduced or no T cell response, and reduced or no B cell response to a population of low immune cells.

In some embodiments, the low immunity cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof. In certain embodiments, the low immunity cell further comprises a genomic modification of the CD142 gene.

In some embodiments, the population of low immunity cells comprises differentiated cells derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells comprise induced pluripotent stem cells. In some embodiments, the differentiated cells are selected from the group consisting of: cardiac cells, neural cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells (e.g., plasma cells or platelets), and epithelial cells.

In some embodiments, the population of low immunity cells comprises cells derived from primary T cells. In some embodiments, the cells derived from the primary T cells are derived from a T cell pool comprising primary T cells from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) subjects other than the patient. In some embodiments, the primary T cell-derived cells comprise a Chimeric Antigen Receptor (CAR).

In some embodiments, the Chimeric Antigen Receptor (CAR) is selected from the group consisting of: (a) A first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) A second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) A third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and the domain following successful signaling of the CAR induces expression of cytokine genes.

In some embodiments, the antigen binding domain is selected from the group consisting of: (a) The antigen binding domain targets the antigenic properties of the tumor cell; (b) An antigen binding domain that targets an antigenic property of a T cell, (c) an antigen binding domain that targets an antigenic property of an autoimmune or inflammatory disorder; (d) An antigen binding domain that targets an antigenic feature of senescent cells; (e) An antigen binding domain that targets an antigenic feature of an infectious disease; and (f) an antigen binding domain that binds to a cell surface antigen of a cell.

In some embodiments of the CAR, the antigen binding domain is selected from the group consisting of: antibodies, antigen binding portions thereof, scFv and Fab. In some embodiments, the antigen binding domain binds to CD19 or BCMA.

In some embodiments of the CAR, the transmembrane domain comprises one selected from the group consisting of: tcra, tcrβ, tcrζ, cd3ζ, cd3γ, cd3δ, cd3ζ, CD4, CD5, cd8α, cd8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40L/CD154, CD45, CD64, CD80, CD86, OX40/CD134, 4-1BB/CD137, CD154, fcsry, VEGFR2, FAS, the transmembrane region of FGFR2B, and functional variants thereof.

In some embodiments of the CAR, the signaling domain comprises a co-stimulatory domain. In some embodiments, the co-stimulatory domain comprises two different co-stimulatory domains. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation.

As described for the fourth generation CARs, successful signaling of the CAR induces expression of cytokine genes. In some embodiments, the cytokine gene is an endogenous or exogenous cytokine gene to the low immunity cell. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-9, IL-12, IL 18, TNF, IFN-gamma and functional fragments thereof. In some embodiments of the fourth generation CAR, the domain that induces cytokine gene expression following successful signaling of the CAR comprises a transcription factor or functional domain or fragment thereof.

In some embodiments, the CAR comprises a cd3ζ domain or an immune receptor tyrosine-based activation motif (ITAM) or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes. In some embodiments, the CAR comprises (i) an anti-CD 19 scFv; (ii) A CD8 a hinge and a transmembrane domain or functional variant thereof; (iii) a 4-1BB co-stimulatory domain or a functional variant thereof; and (iv) a CD3 zeta signaling domain or a functional variant thereof.

In one aspect, provided herein is a method comprising administering to a patient a population of low immunity cells comprising an exogenous CD47 polypeptide and a reduced expression of MHC class I and/or II human leukocyte antigen, wherein the patient has previously received an allograft.

In some embodiments, the allograft is selected from the group consisting of: allogeneic cell grafts, allogeneic blood transfusion, allogeneic tissue grafts, and allogeneic organ grafts. In some embodiments, the patient exhibits a memory B cell and/or memory T cell response to one or more alloantigens. In some embodiments, the one or more alloantigens comprise human leukocyte antigens.

In some embodiments, the patient exhibits reduced or no immune response to a population of low immunity cells. In some embodiments, the reduced or no immune response is selected from the group consisting of: reduced or no systemic immune response, reduced or no adaptive immune response, reduced or no innate immune response, reduced or no T cell response, and reduced or no B cell response to a population of low immune cells.

In some embodiments, a population of low-immunity cells is administered for at least one week or more after the patient receives the allograft. In certain embodiments, a population of low-immunity cells is administered for at least 1 month or more after the patient receives the allograft.

In some embodiments, the low immunity cells comprise MHC class I and class II human leukocyte antigens that reduce expression. In some embodiments, the low immunity cell comprises an exogenous CD47 polypeptide and a reduced expression amount of B2M and/or CIITA. In some embodiments, the low immunity cell comprises an exogenous CD47 polypeptide and reduced expression levels of B2M and CIITA. In some embodiments, the low immunity cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof. In some embodiments, the low immunity cell further comprises a reduced amount of CD142.

In some embodiments, the antigen binding domain is selected from the group consisting of: (a) The antigen binding domain targets the antigenic properties of the tumor cell; (b) An antigen binding domain that targets the antigenic properties of T cells; (c) The antigen binding domain targets the antigenic properties of autoimmune or inflammatory disorders; (d) An antigen binding domain that targets an antigenic feature of senescent cells; (e) An antigen binding domain that targets an antigenic feature of an infectious disease; and (f) an antigen binding domain that binds to a cell surface antigen of a cell. In some embodiments, the antigen binding domain is selected from the group consisting of: antibodies, antigen binding portions thereof, scFv and Fab. In some embodiments, the antigen binding domain binds to CD19 or BCMA.

In some embodiments of the fourth generation CAR-T that induce cytokine gene expression, the cytokine gene is due to an endogenous or exogenous cytokine gene to the low immunity cell. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-9, IL-12, IL 18, TNF, IFN-gamma and functional fragments thereof.

In some embodiments, the CAR derived from cells of the primary T cell comprises a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof.

In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes. In some embodiments, the CAR comprises (i) an anti-CD 19 scFv; (ii) A CD8 a hinge and a transmembrane domain or functional variant thereof; (iii) a 4-1BB co-stimulatory domain or a functional variant thereof; and (iv) a CD3 zeta signaling domain or a functional variant thereof.

In some embodiments, a population of low immune cells causes a reduced amount of or no immune activation in the patient after administration. In some embodiments, a population of low immune cells causes a reduced amount of systemic TH1 activation or no systemic TH1 activation in the patient after administration. In some embodiments, a population of low immune cells causes immune activation of reduced amounts of Peripheral Blood Mononuclear Cells (PBMCs) or no PBMCs in the patient after administration. In some embodiments, a population of low immune cells causes a reduced amount of donor-specific IgG antibodies or no donor-specific IgG antibodies to the low immune cells in the patient after administration. In some embodiments, a population of low immune cells causes reduced or no IgM and IgG antibody production in the patient after administration to the low immune cells. In some embodiments, a population of low-immunity cells causes a reduced amount of cytotoxic T cell killing or non-cytotoxic T cell killing of the low-immunity cells in the patient after administration. In some embodiments, a population of low immune cells does not trigger a systemic acute cellular immune response in the patient after administration.

In another aspect, provided is a method comprising administering to a patient a population of low immunity cells comprising an exogenous CD47 polypeptide and a reduced expression of MHC class I and/or II human leukocyte antigen, wherein the patient has previously exhibited alloimmunization during pregnancy. In some embodiments, the alloimmunization at pregnancy is Hemolytic Disease (HDFN) in the fetus and neonate, neonatal Alloimmune Neutropenia (NAN), or fetal and neonatal alloimmune thrombocytopenia (FNAIT). In some embodiments, the methods described herein are useful for treating a disorder in a patient.

In many embodiments, the low immunity cells comprise reduced expression of MHC class I and class II human leukocyte antigens. In some embodiments, the low immunity cell comprises an exogenous CD47 polypeptide and a reduced expression amount of B2M and/or CIITA. In some embodiments, the low immunity cell comprises an exogenous CD47 polypeptide and reduced expression levels of B2M and CIITA. In certain embodiments, the low immunity cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof. In certain embodiments, the low immunity cell further comprises a reduced amount of CD142.

In some embodiments, the low immunity cell is a differentiated cell derived from a pluripotent stem cell. In certain embodiments, the pluripotent stem cells comprise induced pluripotent stem cells.

In many embodiments, the differentiated cells are selected from the group consisting of: cardiac cells, neural cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells (e.g., plasma cells or platelets), and epithelial cells.

In some embodiments, the low immunity cells comprise cells derived from primary T cells. In certain embodiments, the cells derived from the primary T cells are derived from a T cell pool comprising primary T cells from one or more (e.g., two or more, three or more, four or more, five or more, ten or more, twenty or more, fifty or more, or one hundred or more) subjects other than the patient.

For the fourth generation CARs that induce cytokine gene expression, in some embodiments, the cytokine gene is due to an endogenous or exogenous cytokine gene to the low immunity cell. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-9, IL-12, IL18, TNF, IFN-gamma and functional fragments thereof. In some embodiments, the domain of the CAR that induces expression of the cytokine gene following successful signaling of the CAR comprises a transcription factor or a functional domain or fragment thereof.

In some embodiments, a population of low immune cells causes a reduced amount of or no immune activation in the patient after administration. In some embodiments, a population of low immune cells causes a reduced amount of systemic TH1 activation or no systemic TH1 activation in the patient after administration. In some embodiments, a population of low immunity cells causes immune activation of reduced amounts of Peripheral Blood Mononuclear Cells (PBMCs) or no PBMCs in the patient after administration. In some embodiments, a population of low immune cells causes a reduced amount of donor-specific IgG antibodies or no donor-specific IgG antibodies to the low immune cells in the patient after administration. In some embodiments, a population of low immune cells causes reduced or no IgM and IgG antibody production in the patient after administration to the low immune cells. In some embodiments, a population of low-immunity cells causes a reduced amount of cytotoxic T cell killing or non-cytotoxic T cell killing of the low-immunity cells in the patient after administration. In some embodiments, a population of low immune cells does not trigger a systemic acute cellular immune response in the patient after administration.

In one aspect, provided herein is a method of treating a susceptible patient having a cell defect comprising administering to the patient a population of cells differentiated from stem cells comprising one or more low immunity modifications.

In another aspect, provided herein is a method of treating a susceptible patient who is a candidate for a cell therapy, comprising administering to the patient a population of cells differentiated from stem cells comprising one or more low immunity modifications.

In one aspect, provided herein is a method comprising administering to a patient who is a candidate for cell therapy a population of cells differentiated from stem cells comprising one or more low immunity modifications, wherein the patient received prior treatment for a disorder or disease.

In one aspect, provided herein is a method of treating a susceptible patient who is a candidate for a cell therapy, comprising administering to the patient a population of cells differentiated from stem cells comprising one or more low immunity modifications, wherein the patient is not administered an immunosuppressant prior to, during, or after administration of the population of cells.

In one aspect, provided herein is a method of treating a patient in need thereof having at least partial organ failure, comprising administering to the patient a population of cells differentiated from stem cells comprising one or more low immunity modifications prior to administering at least partial organ transplant to the patient.

In another aspect, provided herein is a method of administering a tissue or organ transplant to a patient in need thereof, comprising administering to the patient a population of cells differentiated from stem cells comprising one or more low immunity modifications prior to administration of the tissue or organ transplant.

In some embodiments, the patient is a sensitive patient. In certain embodiments, the patient is sensitive to previous pregnancy or previous grafts. In certain embodiments, the prior implant is selected from the group consisting of: cell grafts, blood transfusions, tissue grafts, and organ grafts. In some embodiments, the previous graft is an allograft.

In some embodiments, the previous graft is a graft selected from the group consisting of: chimeras of human origin, modified non-human autologous cells, modified autologous cells, autologous tissues and autologous organs. In some embodiments, the patient is sensitive to one or more alloantigens or one or more autoantigens. In certain embodiments, the patient exhibits a memory B cell and/or memory T cell response against one or more alloantigens or one or more autoantigens.

In some embodiments, the patient has allergy. In certain embodiments, the allergy is allergy hay fever, food allergy, insect allergy, drug allergy, and atopic dermatitis selected from the group consisting of.

In certain embodiments, the population of cells comprises cells that express exogenous CD47 polypeptides and have reduced expression of B2M and/or CIITA. In some embodiments, the population of cells is selected from the group consisting of: cardiac cells, neural cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, and Chimeric Antigen Receptor (CAR) T cells.

In some embodiments, the patient exhibits reduced or no immune response to the population of cells. In some embodiments, the immune response is reduced compared to the immune response in a patient or a control subject administered a population of "wild-type" cells. In some embodiments, the reduced or no immune response to the population of cells is exhibited selected from the group consisting of: reduced or no systemic immune response, reduced or no adaptive immune response, reduced or no innate immune response, reduced or no T cell response, and reduced or no B cell response. In the illustrated embodiment, the patient presents: a) A reduced amount of systemic TH1 activation or no systemic TH1 activation after administration of the population of cells; b) A reduced amount of Peripheral Blood Mononuclear Cells (PBMCs) or no PBMCs following administration of the population of cells; c) Upon administration of the population of cells, a reduced amount of donor-specific IgG antibodies or no donor-specific IgG antibodies to the population of cells; d) Upon administration of the population of cells, reduced amounts of IgM and IgG antibodies are produced or no IgM and IgG antibodies are produced to the population of cells; and/or e) a reduced amount of cytotoxic T cell killing or no cytotoxic T cell killing of the population of cells after administration of the population of cells.

In certain embodiments, the patient is not administered an immunosuppressant prior to administration of the population of cells. In some embodiments, the population of cells is administered for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, or at least 1 month or more after the patient is sensitive.

In some embodiments, the stem cell is a pluripotent stem cell. In certain embodiments, the pluripotent stem cells are induced pluripotent stem cells.

In some embodiments, the cell defect is associated with a neurodegenerative disease or the cell therapy is used to treat a neurodegenerative disease. In certain embodiments, the neurodegenerative disease is selected from the group consisting of: leukodystrophy, huntington's disease, parkinson's disease, multiple sclerosis, transverse myelitis, and pemphigus disease (PMD). In some embodiments, the population of cells comprises cells selected from the group consisting of: glial progenitor cells, oligodendrocytes, astrocytes and dopamine neurons. In certain embodiments, the dopamine neuron is selected from the group consisting of: neural stem cells, neural progenitor cells, immature dopamine neurons, and mature dopamine neurons.

In some embodiments, the cell deficiency is associated with diabetes or the cell therapy is used to treat diabetes. In certain embodiments, the population of cells is a population of pancreatic islet cells, including pancreatic beta islet cells. In some embodiments, the pancreatic islet cells are selected from the group consisting of: pancreatic islet progenitor cells, immature pancreatic islet cells, and mature pancreatic islet cells.

In certain embodiments, the cell deficiency is associated with a cardiovascular disorder or disease or the cell therapy is used to treat a cardiovascular disorder or disease. In some embodiments, the population of cells is a population of cardiomyocytes.

In some embodiments, the cell defect is associated with a vascular disorder or disease or the cell therapy is used to treat a vascular disorder or disease. In some embodiments, the population of cells is a population of endothelial cells.

In some embodiments, the cell deficiency is associated with autoimmune thyroiditis or the cell therapy is used to treat autoimmune thyroiditis. In some embodiments, the population of cells is a population of thyroid progenitor cells.

In certain embodiments, the cell deficiency is associated with a liver disease or the cell therapy is used to treat a liver disease. In some embodiments, the liver disease comprises cirrhosis of the liver.

In some embodiments, the population of cells is a population of hepatocytes or hepatic progenitors. In certain embodiments, the cellular defect is associated with a corneal disease or the cell therapy is used to treat a corneal disease. In some embodiments, the corneal disease is Fuchs dystrophy or congenital genetic endothelial dystrophy. In some embodiments, the population of cells is a population of corneal endothelial progenitor cells or corneal endothelial cells.

In some embodiments, the cell deficiency is associated with kidney disease or the cell therapy is used to treat kidney disease. In some embodiments, the population of cells is a population of kidney precursor cells or kidney cells.

In certain embodiments, cell therapy is used to treat cancer. In some embodiments, the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer. In some embodiments, the population of cells is a population of Chimeric Antigen Receptor (CAR) T-cells.

In some embodiments, the prior treatment does not comprise the population of cells. In certain embodiments, the population of cells is administered to treat the same condition or disease previously treated. In some embodiments, the population of cells exhibits an enhanced therapeutic effect on the treatment of a disorder or disease in a patient as compared to prior treatments. In certain embodiments, the population of cells exhibits a longer therapeutic effect on the treatment of a disorder or disease in a patient than prior treatments. In some embodiments, the population of cells is administered to treat a condition or disease that is different from the previous treatment. In some embodiments, the prior treatment is therapeutically ineffective. In some embodiments, the patient develops an immune response to prior treatment.

In some embodiments, the prior treatment comprises administering a population of therapeutic cells comprising a suicide gene safety switch system, and the immune response occurs in response to activation of the suicide gene safety switch system.

In some embodiments, the prior treatment comprises a mechanically assisted treatment. In an exemplary embodiment, the mechanically assisted treatment comprises hemodialysis or ventricular assist devices.

In some embodiments, the tissue and/or organ graft or partial organ graft is selected from the group consisting of: heart grafts, lung grafts, kidney grafts, liver grafts, pancreas grafts, intestinal grafts, stomach grafts, cornea grafts, bone marrow grafts, vascular grafts, heart valve grafts, bone grafts, partial lung grafts, partial kidney grafts, partial liver grafts, partial pancreas grafts, partial intestinal grafts and/or partial cornea grafts. In some embodiments, the population of cells is administered for treating a cell defect in a tissue or organ selected from the group consisting of: heart, lung, kidney, liver, pancreas, intestine, stomach, cornea, bone marrow, blood vessels, heart valves and/or bones.

In some embodiments, the tissue or organ graft is an allograft graft. In certain embodiments, the tissue or organ graft is an autograft graft. In some embodiments, the population of cells is administered to treat a cell defect in a tissue or organ and the tissue or organ transplant is a replacement for the same tissue or organ.

In certain embodiments, the population of cells is administered to treat a cellular defect in a tissue or organ and the tissue or organ transplant is a replacement for a different tissue or organ. In some embodiments, the organ transplant is a kidney transplant and the population of cells is a population of pancreatic beta islet cells. In the illustrated embodiment, the patient has diabetes.

In another aspect, provided herein is a method comprising administering to a patient a population of low immunity cells. In this method, the hypoimmunity cells each comprise: a) An exogenous polynucleotide inserted into a genomic locus comprising a harbour locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus; b) Exogenous CD47 polypeptide; and c) reducing the expression of MHC class I and/or II human leukocyte antigens.

In one aspect, provided herein are methods comprising administering to a patient a dosing regimen. In this method, the dosing regimen comprises a) a first administration comprising a therapeutically effective amount of low immunity cells; b) During recovery; and c) a second administration comprising a therapeutically effective amount of low immunity cells; wherein the low immunity cells each comprise an exogenous polynucleotide inserted into a genomic locus comprising a B2M gene locus, a CIITA gene locus, a TRAC gene locus or a TRB gene locus, and wherein the low immunity cells each comprise an exogenous CD47 polypeptide and an MHC class I and/or II human leukocyte antigen that reduces expression.

In one aspect, provided herein is a use of a population of low-immunity cells for treating a disease in a patient, wherein the low-immunity cells each comprise an exogenous polynucleotide inserted into a genomic locus comprising a safe harbor locus, a locus of interest, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus; and wherein the hypoimmune cells each comprise an exogenous CD47 polypeptide and a reduced expression MHC class I and/or II human leukocyte antigen.

In one aspect, provided herein is a method comprising administering to a patient a population of low immunity cells. In this method, the hypoimmunity cells each comprise: a) An exogenous polynucleotide inserted into a genomic locus comprising a harbour locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus; b) Exogenous CD47 polypeptide; and c) reducing the expressed MHC class I and/or II human leukocyte antigens, wherein the patient has previously received an allograft.

In one aspect, provided herein is a method of treating a patient who is a candidate for cell therapy, comprising administering to the patient a population of low immunity cells. In this method, the hypoimmunity cells each comprise: a) An exogenous polynucleotide inserted into a genomic locus comprising a harbour locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus; b) Exogenous CD47 polypeptide; and c) reducing the expression of MHC class I and/or II human leukocyte antigens.

In one aspect, provided herein is a method comprising administering to a population of low-immunity cells of a patient who is a candidate for cell therapy. In this method, the hypoimmunity cells each comprise: a) An exogenous polynucleotide inserted into a genomic locus comprising a harbour locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus; b) Exogenous CD47 polypeptide; and c) lowering the expressed MHC class I and/or II human leukocyte antigens, wherein the patient received prior treatment for the disorder or disease.

In one aspect, provided herein is a method of treating a patient who is a candidate for cell therapy, comprising administering to the patient a population of low-immunity cells, wherein the low-immunity cells each comprise: a) An exogenous polynucleotide inserted into a genomic locus comprising a B2M gene locus, a CIITA gene locus, a TRAC gene locus or a TRB gene locus; b) Exogenous CD47 polypeptide; and c) reducing the expressed MHC class I and/or II human leukocyte antigen, wherein the patient is not administered an immunosuppressant prior to, during or after administration of the population of cells.

In another aspect, provided herein is a method of treating a patient in need thereof having at least partial organ failure, comprising administering to the patient a population of low immunity cells. In this method, the hypoimmunity cells each comprise: a) An exogenous polynucleotide inserted into a genomic locus comprising a harbour locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus; b) Exogenous CD47 polypeptide; and c) reducing the expressed MHC class I and/or II human leukocyte antigens, wherein a population of low immunity cells is administered prior to administering at least a portion of the organ transplant to the patient.

In yet another aspect, provided herein is a method for administering a tissue or organ transplant to a patient in need thereof, comprising administering to the patient a population of low immunity cells. In this method, the hypoimmunity cells each comprise: a) An exogenous polynucleotide inserted into a genomic locus comprising a harbour locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus; and b) an exogenous CD47 polypeptide, wherein a population of low immunity cells is administered prior to administration of the tissue or organ graft.

In another aspect, provided herein is a method for administering to a patient a population of low immunity cells. In this method, the hypoimmunity cells each comprise: a) A genetic modification comprising an exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR) inserted into a genomic locus comprising a harbour locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus; b) Exogenous CD47 polypeptide; and c) reducing the expression of MHC class I and/or II human leukocyte antigens.

In another aspect, provided herein is a method of treating cancer in a patient in need thereof, comprising administering to the patient a population of low immunity cells. The hypoimmune cells each comprise: a) An exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR) inserted into a genomic locus comprising a harbour locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus or a TRB locus; b) Exogenous CD47 polypeptide; and c) reducing the expression of MHC class I and/or II human leukocyte antigens.

In some embodiments, the low immunity cell comprises an additional exogenous polynucleotide encoding an exogenous CD47 polypeptide. In certain embodiments, the additional exogenous polynucleotide is i) located at a genomic locus different from the genomic locus in (a); or ii) located at the same genomic locus as in (a).

In another aspect, provided herein are methods comprising administering to a patient a population of low immunity cells. In this method, the hypoimmunity cells each comprise: a) A first exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR) inserted into a first genomic locus; and B) a second exogenous polynucleotide encoding a CD47 polypeptide inserted into a second genomic locus, wherein the low immunity cell exhibits reduced expression of MHC class I and/or II human leukocyte antigens, wherein the first and second genomic loci are each a safe harbor locus, a locus of interest, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus.

In one aspect, provided herein is a method of treating cancer in a patient in need thereof, comprising administering to the patient a population of low immunity cells. In this method, the hypoimmunity cells each comprise: a) A first exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR) inserted into a first genomic locus; and B) a second exogenous polynucleotide encoding a CD47 polypeptide inserted into a second genomic locus, wherein the low immunity cell exhibits reduced expression of MHC class I and/or II human leukocyte antigens, wherein the first and second genomic loci are each a safe harbor locus, a locus of interest, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus.

In some embodiments, the first and second genomic loci are the same. In certain embodiments, the first and second genomic loci are different. In some embodiments, the low immunity cells each further comprise a third exogenous polynucleotide inserted into a third genomic locus. In some embodiments, the third genomic locus is identical to the first or second genomic locus. In some embodiments, the third genomic locus is different from the first and/or second genomic loci.

In some embodiments, the safe harbor or target locus is selected from the group consisting of: CCR5 gene locus, CXCR4 gene locus, PPP1R12C (also known as AAVS 1) gene, albumin gene locus, SHS231 gene locus, CLYBL gene locus, ROSA26 gene locus, CD142 gene locus, MICA gene locus, MICB gene locus, LRP1 gene locus, HMGB1 gene locus, ABO gene locus, RHD gene locus, FUT1 gene locus, PDGFRa gene locus, OLIG2 gene locus, GFAP gene locus and KDM5D gene locus. In certain embodiments, the CCR5 gene locus is exons 1 to 3, introns 1 to 2, or the coding sequence (CDS) of the CCR5 gene. In some embodiments, the PPP1R12C gene locus is intron 1 or intron 2 of the PPP1R12C gene. In some embodiments, the CLYBL gene locus is intron 2 of the CLYBL gene. In certain embodiments, the ROSA26 gene locus is intron 1 of the ROSA26 gene. In some embodiments, the harbor locus of interest is the SHS231 locus. In some embodiments, the CD142 gene locus is the CDs of the CD142 gene. In certain embodiments, the MICA gene locus is the CDS of the MICA gene. In some embodiments, the MICB gene locus is the CDS of the MICB gene. In some embodiments, the B2M gene locus is the CDS of the B2M gene. In an exemplary embodiment, the CIITA gene locus is the CDS of the CIITA gene. In certain embodiments, the TRAC gene locus is the CDS of the TRAC gene. In some embodiments, the TRB gene locus is the CDS of the TRB gene.

In certain embodiments, the exogenous polynucleotide is operably linked to a promoter.

In some embodiments, the low immunity cell is a differentiated cell derived from a pluripotent stem cell. In some embodiments, the pluripotent stem cells comprise induced pluripotent stem cells.

In certain embodiments, the differentiated cells are selected from the group consisting of: pancreatic beta islet cells, glial progenitor cells, heart cells, nerve cells, endothelial cells, B cells, retinal pigment epithelial cells, liver cells, thyroid cells, skin cells, blood cells (e.g., plasma cells or platelets), and epithelial cells. In some embodiments, the differentiated cell is a T cell.

In some embodiments, the low immunity cell is derived from a primary T cell. In certain embodiments, the low immunity cell is a T cell derived from a pluripotent stem cell. In some embodiments, the low immunity cell is derived from a primary T cell. In some embodiments, the exogenous polynucleotide encodes a Chimeric Antigen Receptor (CAR).

In an exemplary embodiment, the Chimeric Antigen Receptor (CAR) is selected from the group consisting of: a) A first generation CAR comprising at least an antigen binding domain, a transmembrane domain, and a signaling domain; b) A second generation CAR comprising at least one antigen binding domain, a transmembrane domain, and at least two signaling domains, c) a third generation CAR comprising at least one antigen binding domain, a transmembrane domain, and at least three signaling domains; and d) a fourth generation CAR comprising at least one antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of cytokine genes following successful signaling of the CAR.

In some embodiments, at least one antigen binding domain is selected from the group consisting of: a) An antigen binding domain that targets the antigenic properties of tumor cells; b) An antigen binding domain that targets the antigenic properties of T cells; c) The antigen binding domain targets the antigenic properties of autoimmune or inflammatory disorders; d) An antigen binding domain that targets an antigenic feature of senescent cells; e) An antigen binding domain that targets an antigenic feature of an infectious disease; and f) an antigen binding domain that binds to a cell surface antigen of a cell.

In certain embodiments, at least one antigen binding domain is selected from the group consisting of: antibodies, antigen binding portions thereof, scFv and Fab. In some embodiments, the CAR is a bispecific CAR comprising two antigen binding domains that bind two different antigens. In some embodiments, at least one antigen binding domain binds to an antigen selected from the group consisting of: CD19, CD22 and BCMA. In certain embodiments, the bispecific CAR binds to CD19 and CD22.

In some embodiments, the transmembrane domain of the CAR comprises a transmembrane region selected from the group consisting of: the transmembrane region from tcra, tcrβ, tcrζ, cd3ε, cd3γ, cd3δ, cd3ζ, CD4, CD5, cd8α, cd8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40L/CD154, CD45, CD64, CD80, CD86, OX40/CD134, 4-1BB/CD137, CD154, fcsry, VEGFR2, FAS, FGFR2B, and functional variants thereof.

In certain embodiments, the signaling domain of the CAR comprises a co-stimulatory domain. In certain embodiments, the co-stimulatory domain comprises two different co-stimulatory domains. In some embodiments, the co-stimulatory domain enhances cytokine production, CAR-T cell proliferation, and/or CAR-T cell persistence during T cell activation. In some embodiments, the cytokine gene is an endogenous or exogenous cytokine gene to the low immunity cell. In some embodiments, the cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is selected from the group consisting of: IL-1, IL-2, IL-9, IL-12, IL 18, TNF, IFN-gamma and functional fragments thereof. In certain embodiments, the domain that induces cytokine gene expression following successful signaling of the CAR comprises a transcription factor or functional domain or fragment thereof.

In some embodiments, the CAR comprises a CD3 zeta (cd3ζ) domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof. In certain embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; and (iii) a 4-1BB domain or a CD134 domain or a functional variant thereof. In some embodiments, the CAR comprises (i) a cd3ζ domain or an immunoreceptor tyrosine-based activation motif (ITAM) or a functional variant thereof; (ii) a CD28 domain or a functional variant thereof; (iii) A 4-1BB domain or a CD134 domain or a functional variant thereof; and (iv) cytokine or co-stimulatory ligand transgenes. In certain embodiments, the CAR comprises (i) an anti-CD 19 scFv; (ii) A CD8 a hinge and a transmembrane domain or functional variant thereof; (iii) a 4-1BB co-stimulatory domain or a functional variant thereof; and (iv) a CD3 zeta signaling domain or a functional variant thereof.

In some embodiments, the low immunity cell comprises an endogenous T cell receptor that reduces expression. In some embodiments, the low immunity cells comprise reduced expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA 4) and/or programmed cell death (PD 1). In certain embodiments, the low immunity cell comprises a programmed cell death ligand 1 (PD-L1) that increases expression.

In some embodiments, the patient is sensitive to one or more alloantigens. In some embodiments, the patient is susceptible to prior pregnancy or prior allograft. In certain embodiments, the one or more alloantigens comprise human leukocyte antigens.

In some embodiments, the patient exhibits a memory B cell and/or memory T cell response to one or more alloantigens. In certain embodiments, the allograft is selected from the group consisting of: allogeneic cell grafts, allogeneic blood transfusion, allogeneic tissue grafts, and allogeneic organ grafts.

In some embodiments, the patient exhibits reduced or no immune response to the population of cells. In certain embodiments, the cell response to the population exhibits reduced or no immune response selected from the group consisting of: reduced or no systemic immune response, reduced or no adaptive immune response, reduced or no innate immune response, reduced or no T cell response, and reduced or no B cell response.

In some embodiments, the patient presents: a) A reduced amount of systemic TH1 activation or no systemic TH1 activation after administration of the population of cells; b) A reduced amount of Peripheral Blood Mononuclear Cells (PBMCs) or no PBMCs following administration of the population of cells; c) Upon administration of the population of cells, a reduced amount of donor-specific IgG antibodies or no donor-specific IgG antibodies to the population of cells; d) Upon administration of the population of cells, reduced amounts of IgM and IgG antibodies are produced or no IgM and IgG antibodies are produced to the population of cells; and/or e) a reduced amount of cytotoxic T cell killing or no cytotoxic T cell killing of the population of cells after administration of the population of cells.

In some embodiments, the disorder is cancer or cell therapy is used to treat cancer. In some embodiments, the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

V. examples

Example 1: different speciesHuman B2M in the seed transplantation study ^indel/indel 、CIITA ^indel/indel CD47tg induced pluripotent stem cell

To study the effect of reducing MHC I and MHC II expression and increasing CD47 expression on cell grafts, human B2M was used ^indel/indel 、CIITA ^indel/indel CD47tg induced pluripotent stem cells (HIP cells) were transplanted into rhesus (non-human primate or NHP) recipients (xenografts).

Study design and administration. Eight NHPs (F/M, 2-3kg,12-36 months old) were randomized into two groups (n=4) for blind administration of wild-type or HIP cells. According to IACUC approved protocol, each NHP was given subcutaneously 4 times to 10 times on the back ⁷ Individual wild-type or HIP cells. The characteristics of the human wild-type iPSC and the human HIP iPSC are shown in fig. 14. Blood draw analysis prior to injection ("pre Tx" or day 0), at day 7, 13, 25, etc. after injection. Firefly luciferases, which are also expressed transgenically by wild-type and HIP cells, are used in bioluminescence imaging (BLI), and cell viability is monitored by BLI. The study designs and results are shown in figures 1A-1F, 2A, 2B, 3A, 3B, 4A to 4C, 5A to 5C, 6A to 6C, and 7A to 7C.

No systemic immune response was observed in NHPs that received xenogenic HIP cells after initial injection, which showed increased T cell activation, igM and IgG amounts, and donor specific IgM and IgG compared to NHPs that injected wild type cells. To determine if HIP cells can be re-administered in a similar lack of immune activation, NHPs were re-injected with the same cell type (wild type or HIP) as the second injection between day 118 and day 123 after the first injection. As before, blood was drawn before reinjection ("pre Tx" or day 0) and on days 7 and 13 after reinjection (125 and 131 days after the first injection, respectively) for analysis, and cell viability was monitored by BLI. Notably, no systemic immune response was observed in animals re-injected with xenogenic HIP cells, whereas animals re-injected with wild type cells showed systemic immune activation. Although no systemic immune activation was seen in animals administered HIP cells, cells failed to survive during 13 days of initial or second dosing (BLI < 5% of initial dose), apparently due to local xenogeneic response and response to vehicle (Matrigel). These results indicate that HIP cells can escape multiple doses of immune recognition and activation.

To determine if HIP cells can escape the preformed immune response, four NHPs initially administered with two doses of wild-type cells were transplanted with HIP cells and vice versa (cross administration). HIP or wild-type cells were injected subcutaneously into animals between day 118 and 123 after the second injection (day 241 after the initial injection). As before, blood was drawn for analysis 48 days before and 7 and 13 days thereafter (248 and 254 days after the first injection, respectively) and cell viability was monitored by BLI.

T cell activation. T cell activation was measured by the Elispot assay for wild type and HIP human iPSC administered animals. For the unidirectional Elispot assay, recipient PBMCs were isolated from rhesus macaques 48 days before and 7 days after the third injection (cross administration) and 13 days. T cells were purified from PBMCs by CD3 MACS-sorting (Miltenyi) and used as responder cells. Donor cells (wild-type or HIP cells) were mitomycin treated (50 μg/mL for 30 min, sigma) and used as stimulator cells. 1X 10 ⁵ Individual stimulator cells and 5×10 cells ⁵ The recipient responders T-cells were incubated for 36 hours and the IFN- γ spot frequency was calculated using an Elispot disk reader. For animals administered wild-type cells after the first two injections of HIP cells, the observed Elispot activity was highest at day 7 after the crossover injection (FIGS. 1A to 1F). These results indicate that following injection of wild-type cells, systemic TH1 activation and acute cellular immune response, the previously injected HIP cells were not immunosuppressed. In contrast, animals injected with HIP cells after the first two injections of wild type cells (cross injection) had an Elispot activity on day 0 comparable to that of the original TH1 cells, indicating no systemic TH1 activation or cellular immune response to the modified cells, even in animals with preformed immune response to wild type xenogenesis (FIGS. 1A-1F).

Donor-specific antibody activity. Donor-specific antibodies generated from animals cross-injected with wild-type and HIP cells were also assayed. Serum from recipient monkeys was heated to 56 ℃ for 30 minutesComplement removal. Equal amounts of serum and wild-type or HIP cell suspensions (5X 10 ⁶ Individual cells/mL) were incubated at 4℃for 45 min. Cells were labeled with FITC-conjugated goat anti-IgM (BD Bioscience) or anti-IgG and analyzed by flow cytometry (BD Bioscience).

Donor specific reactivity was observed to be higher than before on days 7 and 13 after cross injection of wild type cells in animals previously administered HIP cells, igM declined from day 7 to day 13 consistent with isotype switching (data not shown). In contrast, no donor-specific IgM binding was observed in animals previously receiving two wild-type cell injections to which HIP cells were administered (data not shown). An increase in donor-specific reactivity was observed on day 13 after cross-injection of wild-type cells in animals previously administered HIP cells, igG increased from day 7 to day 13, and then decreased from day 13 to day 75, consistent with isotype switching (fig. 3A-3B). In contrast, no donor-specific IgG binding was observed in animals administered HIP cells that had previously received two wild-type cell injections (fig. 2A and 2B) on days 7, 13 and 75.

Batch antibody production. Total antibody production in animals receiving wild-type or HIP cell cross-injections was assayed using IgM and IgG ELISA kits (Abcam). After removal of unbound proteins by washing, anti-IgM or anti-IgG antibodies conjugated to horseradish peroxidase (HRP) were added. These enzyme-labeled antibodies form complexes with previously bound IgM or IgG. The enzyme bound to the immunoadsorbent is assayed by the addition of chromogenic substrate 3,3', 5' -tetramethyl-benzidine (TMB). After two administrations of HIP cells, a dramatic increase in total IgM and IgG was observed in animals cross-administered with wild-type cells, maximum IgM production was observed on day 7 and maximum IgG production on day 13, indicating isotype switching (fig. 4A to 4C and 6A to 6C).

Remarkably, in animals cross-administered HIP cells after the two injections of wild-type cells, no increase in total IgM or IgG was observed at any time point (fig. 5A to 5C and 7A to 7C).

Some IgG was observed prior to HIP injection, possibly residual production from previous wild-type administration (fig. 7A to 5C). Taken together, these results indicate a near complete lack of humoral immune response to HIP cells.

NK cell killing. Systemic innate immunity of NK cells was also assayed in animals cross-injected with wild-type or HIP cells. NK cell killing assays were performed on the XCELLIGENCE MP platform (ACEA Biosciences). 96 well E-disc (ACEA Biosciences) was coated with collagen (Sigma-Aldrich), and 4X 10 ⁵ The individual wild-type or HIP cells were seeded in 100. Mu.l of cell-specific medium. After the cell index value reached 0.7, the ratio was set at 1: e of 1: t ratio rhesus NK cells isolated from treated animals were added with or without 1ng/ml rhesus IL-2 (MyBiosource, san Diego, calif.). As a killing control, cells were treated with 2% triton x 100. The lack of killing of wild-type or HIP cells by stimulated or unstimulated NK cells indicates that CD47 expression on HIP cells is effective in protecting from NK cells and macrophages in the absence of HLA I and HLA II. (see Deuse et al, 2019, nat. Biotechnol., 37:252-258). As shown in fig. 8A to 8C, no NK cell killing was observed after administration of the first dose of HIP cells to wild-type NHP (fig. 8C), nor after re-administration of HIP cells to wild-type NHP (fig. 8C). Although HLA I/HLA II (e.g. MHC editing)) was directed against HIP cells, lack of NK cell killing was also observed after cross-injection of HIP cells to wild-type NHPs with pre-existing immunity (fig. 8D and 8E).

Survival of transplanted cells. Although no systemic immune response was observed in animals cross-administered with human HIP cells, it is likely that the cells did not survive due to local xenogenic responses. Histopathological analysis of the plugs removed from animals for the previous wild-type and HIP injections showed evidence of neutrophil infiltration or fibrin (as an indicator of neutrophils already in the area) as well as foreign body response and type IV hypersensitivity to vehicle indicating xenogenic and allergic responses to human cells, respectively to vehicle. Allergic and foreign body responses to vehicle were confirmed by additional vehicle-only (cell-free) control monkeys, which demonstrated similar histopathological features.

This example demonstrates that HIP cells can be administered to subjects with pre-existing systemic alloimmune responses without eliciting a new systemic immune response.

Example 2: human B2M in allograft crossover studies ^indel/indel 、CIITA ^indel/indel CD47tg Induced Pluripotent Stem Cells (iPSCs) and wild type IPSCs

This example describes an allograft crossover study comparing transplanted human B2M ^indel/indel 、CIITA ^indel ^/indel Effect of CD47tg induced pluripotent stem cells (HIP iPSC) and wild-type iPSC to rhesus (non-human primate or NHP) recipients. In one set of crossover studies, wild-type ipscs were transplanted subcutaneously (s.c,) to the back of recipient animals, and after about 6 weeks, HIP ipscs were transplanted in an adjacent location. In a second set of crossover studies, HIP ipscs were s.c. transplanted to the back of recipient animals, and after about 6 weeks, wild-type ipscs were transplanted to adjacent sites. The presence of the implanted cells and their progeny is monitored.

The data show that HIP ipscs were not detected by the immune system of the sensitive NHP recipient (the NHP recipient initially transplanted with wild-type ipscs), thus avoiding immune rejection. Even if the recipient has a functional immune system, the implanted HIPiPSC is able to escape the recipient's immune response. In addition, the NHP recipient initially transplanted with HIP iPSC had an immune response to the wild-type iPSC following transplantation.

A. Method of

Gene editing of human iPSC over-expressed rhesus CD 47. Human iPSC B2M ^indel/indel 、CIITA ^indel/ Rhesus CD47 tg cells (also referred to as HIP iPSC or HIP cells) are cultured using standard human iPSC cell culture methods recognized by those of ordinary skill in the art to which the invention pertains. The characteristics of the rhesus wild-type iPSC and the rhesus HIP iPSC are shown in fig. 15.

Culturing rhesus iPSC cells. Rhesus ipscs were cultured using standard rhesus iPSC cell culture methods approved by the general knowledgeable in the art of the invention.

Luciferase transduction of hiPSC. hipscs (i.e., HIP ipscs and wild-type ipscs) were infected by lentiviral particles expressing the luciferase II gene under the expression control of a constitutively active promoter (i.e., CAG promoter). Luciferase expression by infected cells was confirmed using standard commercially available luciferase assays.

Ipscs transplanted to non-human primates were prepared. ipscs were resuspended in standard media including pro-survival cocktails (i.e., cocktails including apoptosis protease inhibitor, bcL-xL, IGF-1, pinacoidil, and cyclosporin a). Cells were loaded into a syringe for injection.

Intramuscular iPSC injection in rhesus macaques. Animals are sedated by Intramuscular (IM) injection of a fast-acting anesthetic (i.e., a combination of tilitamine and zolazepam), preferably not in the legs receiving the cell implant. Once anesthetized, the animal's legs are shaved at the catheter and cell implantation site. Blood samples were collected from femoral veins by percutaneous venipuncture. The catheter is placed into the saphenous vein (preferably not in the leg receiving the cell implant). The cell implantation area, i.e., the anterior surface of the thigh or quadriceps, was surgically washed with alternating doherticidine gluconate/ethanol washes, and finally completed with doherticidine gluconate.

An incision was made in the skin on the medial anterior side of the quadriceps femoris muscle of the animal. By pinching the isolated quadriceps, while ipscs are injected in a starburst pattern, the injected cells are injected into multiple locations within the pattern. The incision is closed with a suture and the injection area is marked for future reference.

Fluorescein was injected into recipient animals through pre-placed intravenous catheters for fluorescein infusion. Once the animal's vital signs, such as heart rhythm, return to normal, the injection area is imaged by bioluminescence imaging (BLI). BLI monitors cell viability. Over time, quantitative bioluminescence imaging data are represented as BLI images and BLI signals.

Transplantation of HIP iPSC

As shown in fig. 9A, the allogeneic HIP rhesus ipscs were transplanted into the left leg of the rhesus recipient. The HIP cells do not elicit an immune response in the recipient. The implanted cells were examined at the injection site at least 6 weeks after implantation. Fig. 9B shows immunohistochemical staining of the left leg implanted with HIP iPSC 6 weeks after implantation. Fig. 9B shows staining of Smooth Muscle Actin (SMA) representing blood vessels, and shows luciferase of transplanted HIP ipscs.

In addition, fig. 13C shows BLI images of a similar study used to monitor the presence of transplanted allogeneic HIP rhesus ipscs in the left leg of allogeneic rhesus recipients. The transplanted cells and their progeny were found at the injection site at least 9 weeks after the initial transplantation. HIP ipscs did not elicit a significant immune response in rhesus recipients, as cells persisted for at least 9 weeks after transplantation.

C. Crossover study: administration of wild-type iPSC followed by HIP iPSC in the same NHP

In a cross-over study of wild-type iPSC to HIP iPSC, an allogeneic rhesus wild-type iPSC was transplanted into the left leg of a rhesus recipient. The transplanted rhesus wild-type iPSC population was substantially reduced on day 7 post-transplantation (100% to 6.8%; fig. 10). Only 10% of the transplanted population was detected 2 weeks after transplantation, while only 1.4% of the population was maintained 3 weeks after transplantation. 4 and 5 weeks after the transplantation, no transplanted cells were found at the injection site. Thus, rhesus recipients appear to become sensitive. In the cross arm of the study, allogeneic HIP rhesus ipscs were injected into the right leg of the sensitive rhesus recipient 5 weeks after the initial wild-type iPSC graft (also referred to as day 0 (d 0) crossover).

On day 0 of cross-transplantation, transplanted allogeneic HIP rhesus ipscs were detected at the injection site (fig. 10, bottom row). On day 7 of cross-transplantation (d 7), 69.2% of transplanted HIPiPSCs were detected. In addition, 48.1% of the cells remained at 2 weeks after cross-transplantation. Thus, in the initial arm of the study, recipient animals elicited an immune response to wild-type ipscs, while in the cross arm, HIP ipscs persisted in sensitive recipient animals.

Fig. 11 shows the results of another crossover study of wild-type iPSC to HIP iPSC. The transplanted rhesus wild-type ipscs elicit an immune response in the initial recipient. Specifically, only 10.2% of the transplanted wild-type ipscs were detected on day 7 post-transplantation. At 5 weeks after initial transplantation of the rhesus wild-type iPSC (also referred to as cross-transplanted d 0), the HIP rhesus iPSC was transplanted into the right leg of the now-sensitized rhesus recipient. Transplanted HIPiPSC was detected at the injection site (FIG. 11, bottom row). On day 7 after cross-transplantation, 28.8% of the transplanted cells and their progeny were located at the injection site. 3 weeks after cross-transplantation, the detected population was approximately 32.9% of the transplanted HIPiPSC.

D. Crossover study: HIPiPSC was administered in the same NHP followed by wild type iPSC

In the HIP iPSC to wild-type iPSC crossover study, the allogeneic HIP iPSC was transplanted into the left leg of rhesus recipients (fig. 12). Transplanted HIPiPSC and its progeny were detected at the injection site to at least 9 weeks after transplantation. 5 weeks after implantation, there was about 112% of the initial population of HIPiPSCs and their progeny, while at 7 weeks, 202.4% of HIPiPSCs and their progeny were present. 154.8% and 178.6% HIPiPSC and their progeny were present at 8 weeks and 9 weeks, respectively. HIP ipscs were found in the recipient for at least 9 weeks after the initial graft.

At week 6 after initial transplantation of HIP ipscs (also referred to as day 0 cross-transplantation), the allogeneic rhesus wild-type ipscs were transplanted into the right leg of the rhesus recipient. Transplanted wild-type ipscs were detected at the injection site (fig. 12, bottom row). On day 7 after cross-transplantation, none of the transplanted cells and their progeny were located at the injection site. No luciferase signal was detected. In contrast, at 7 weeks post initial transplantation of HIP ipscs, there was approximately 202.4% of the initial transplanted HIP iPSC population and its progeny in the rhesus recipient left leg.

The results of the above series of crossover studies showed that HIPiPSC was able to evade the immune system of sensitive NHP recipients (NHP recipients initially transplanted with wild type iPSC) and thus, HIPiPSC could avoid immune rejection. In addition, recipients of the initial transplantation of HIP iPSCs developed an immune response to the wild type iPSCs of the subsequent transplantation. See, for example, fig. 13A and 13B. Even if the recipient has a functional immune system, the implanted HIPiPSC is able to escape the immune response.

Example 3: human B2M using safe harbor site ^indel/indel 、CIITA ^indel/indel Expression of exogenous CD47 in CD47tg Induced Pluripotent Stem Cells (iPSCs)

This example describes characterization in human B2M ^indel/indel 、CIITA ^indel/indel Research on expression of exogenous CD47 expression in CD47tg induced pluripotent stem cells (ipscs), wherein a polynucleotide encoding exogenous CD47 is inserted into the safe harbor site of the iPSC.

B2M ^indel/indel 、CIITA ^indel/indel Induced pluripotent stem cells (ipscs) were generated using standard CRISPR/Cas9 gene editing techniques. The HDR donor plasmid encoding human CD47 was introduced into B2M in an expression cassette driven by the CAG or EF1 alpha promoter and flanked by three homology arms of 1kb at the safe harbor sites (AAVS 1, CLYBL or CCR 5) ^indel/indel 、CIITA ^indel/indel iPSC。

Target integration at safe harbor site CD47 is achieved using standard CRISPR/Cas9 gene editing techniques to mediate homology directed repair. The following batch editing strains were generated:

·CAG-CD47 AAVS1

·CAG-CD47_CLYBL

·CAG-CD47_CCR5

·EF1α-CD47_AAVS1

·EF1α-CD47_CLYBL

·EF1α-CD47_CCR5。

monoclonal strains from the bulk editing strain were performed. The clone was evaluated for copy number and plasmid insertion and PCR genotyping was performed using standard techniques to verify the correct position for integration into the safe harbor site. Clones assessed by genome were amplified and subjected to a clonal selection assay to reduce to 2 or 3 clones at each safe harbor site. B2M pair using flow cytometry ^indel/indel 、CIITA ^indel/indel Assessment of CD47 expression of CD47tg clone.

As shown in FIG. 16, B2M in which the CD47 transgene was inserted into each of the three harbor sites ^indel/indel 、CIITA ^indel ^/indel CD47tg exhibits enhanced CD47 expression at-30 to 200 fold over endogenous amounts. It was also observed that CD47 was stably expressed by the CAG promoter from several safe harbor sites in iPSC(see FIGS. 17 and 18). Further evaluation of B2M using the methods described above ^indel ^/indel 、CIITA ^indel/indel CD47tg iPSC was protected from systemic innate immunity. As shown in FIG. 19, B2M comprising CD47 transgene inserted into safe harbor site ^indel/indel 、CIITA ^indel/indel CD47tg iPSC stably expressed CD47 in sufficient quantity to protect against NK and macrophage killing.

All headings and chapter names are for clarity and reference purposes only and should not be construed as limiting in any way. For example, those of ordinary skill in the art to which the invention pertains will appreciate the usefulness of appropriately combining the various aspects from the different headings and chapters in accordance with the spirit and scope of the techniques described herein.

All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Many modifications and variations of this application can be made without departing from its spirit and scope, as will be apparent to those skilled in the art to which the invention pertains. The specific embodiments and examples described herein are offered by way of illustration only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Sequence listing

<110> sana Biotechnology Co., ltd (Sana Biotechnology, inc.)

<120> methods of treating sensitive patients with low immunity cells and related methods and compositions

<130> 122864-01-5044

<150> 63/065,342

<151> 2020-08-13

<150> 63/136,137

<151> 2021-01-11

<150> 63/151,628

<151> 2021-02-19

<150> 63/175,030

<151> 2012-04-14

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Claims

1. A method of treating a patient in need thereof comprising administering a population of low immunity cells, wherein the low immunity cells comprise a first exogenous polynucleotide encoding CD47 and

(I) One or more of the following:

a. major Histocompatibility Complex (MHC) class I and/or II human leukocyte antigens that reduce expression;

b. reduced expression of MHC class I and class II human leukocyte antigens;

c. reduced expression of beta-2-microglobulin (B2M) and/or MHC class II transducins (CIITA); and/or

d. B2M and CIITA with reduced expression;

wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification;

(II) wherein the patient is a sensitive patient, wherein the patient:

i. sensitive to one or more alloantigens;

sensitive to one or more autoantigens;

sensitive due to previous grafts;

Sensitivity due to previous pregnancy;

receiving prior treatment for the disorder or disease; and/or

Is a tissue or organ transplant patient and the low immunity cells are administered prior to, concurrently with and/or after the administration of the tissue or organ transplant.

2. A method of treating a patient in need thereof, comprising administering a population of pancreatic islet cells, wherein the pancreatic islet cells comprise a first exogenous polynucleotide that encodes CD47, and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient, wherein the patient:

i. sensitive to one or more alloantigens;

sensitive to one or more autoantigens;

sensitive due to previous grafts;

Sensitivity due to previous pregnancy;

receiving prior treatment for the disorder or disease; and/or

Is a tissue or organ patient, and the pancreatic islet cells are administered prior to administration of the tissue or organ transplant.

3. A method of treating a patient in need thereof, comprising administering a population of cardiac progenitor cells, wherein the cardiac progenitor cells comprise a first exogenous polynucleotide encoding CD47, and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient, wherein the patient:

i. sensitive to one or more alloantigens;

sensitive to one or more autoantigens;

sensitive due to previous grafts;

sensitivity due to previous pregnancy;

Receiving prior treatment for the disorder or disease; and/or

Is a tissue or organ patient and the cardiac muscle cells are administered prior to the administration of the tissue or organ transplant.

4. A method of treating a patient in need thereof, comprising administering a population of glial progenitor cells, wherein the glial progenitor cells comprise a first exogenous polynucleotide encoding CD47 and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient, wherein the patient:

i. sensitive to one or more alloantigens;

sensitive to one or more autoantigens;

sensitive due to previous grafts;

sensitivity due to previous pregnancy;

Receiving prior treatment for the disorder or disease; and/or

Is a tissue or organ patient, and the glial progenitor cells are administered prior to the administration of the tissue or organ transplant.

5. The method of any one of claims 1 to 4, wherein the patient is a sensitive patient, and wherein the patient exhibits a memory B-cell and/or memory T-cell response to the one or more alloantigens or one or more autoantigens.

6. The method of claim 5, wherein the one or more alloantigens comprise human leukocyte antigens.

7. The method of any one of claims 1 to 6, wherein the patient is a sensitive patient that is sensitive to a previous graft, wherein:

a. the previous graft is selected from the group consisting of: cell grafts, blood transfusions, tissue grafts and organ grafts, the previous grafts being allografts as required; or (b)

b. The previous graft is a graft selected from the group consisting of: chimeric, modified non-human autologous cells, modified autologous cells, autologous tissues and organs of human origin, optionally, the previous graft is an autograft.

8. The method of any one of claims 1 to 6, wherein the patient is a susceptible patient who is susceptible to prior pregnancy, and wherein the patient has previously exhibited an alloimmunity during pregnancy, optionally wherein the alloimmunity during pregnancy is Hemolytic Disease (HDFN) in fetuses and newborns, neonatal Alloimmune Neutropenia (NAN), or fetal and neonatal alloimmune thrombocytopenia (FNAIT).

9. The method of any one of claims 1 to 6, wherein the patient is a susceptible patient susceptible to prior treatment of a disorder or disease, wherein the disorder or disease is different from or the same as the disorder or disease the patient received treatment in any one of claims 1 to 6.

10. The method of any one of claims 1 to 6 or claim 9, wherein the patient receives a prior treatment for a disorder or disease, wherein the prior treatment does not comprise the population of cells, and wherein:

a. administering the population of cells for treating the same disorder or disease as the prior treatment;

b. the population of cells exhibits an enhanced therapeutic effect on treatment of the disorder or disease in the patient compared to the prior treatment;

c. The population of cells exhibits a longer therapeutic effect on the treatment of the disorder or disease in the patient than the prior treatment;

d. the previous treatment is therapeutically effective

e. The previous treatment is treatment-ineffective;

f. the patient develops an immune response to the prior treatment; and/or

g. The population of cells is administered for the treatment of a condition or disease that is different from the previous treatment.

11. The method of claim 10, wherein the prior treatment comprises administering a population of therapeutic cells comprising a suicide gene or a safety switching system, and the immune response occurs in response to activation of the suicide gene or the safety switching system.

12. The method of claim 10, wherein the prior treatment comprises a mechanically assisted treatment, optionally wherein the mechanically assisted treatment comprises hemodialysis or ventricular assist devices.

13. The method of claim 10, wherein the prior treatment comprises an allogeneic CAR-T cell-based therapy or an autologous CAR-T cell-based therapy, wherein the autologous CAR-T cell-based therapy is selected from the group consisting of: bucarba Ji Aolun, sicalico, ai Kaba Ji Weisai, li Jimai Racemosaicism, te Sha Jinlu, descartes-08 or Descartes-11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Cellectis, PBCAR19B or PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics and CYAD-211 from Clyad Oncology.

14. The method of any one of claims 1 to 12, wherein the patient suffers from an allergy, optionally wherein the allergy is an allergy selected from the group consisting of: hay fever, food allergies, insect allergies, drug allergies, and atopic dermatitis.

15. The method of any one of claims 1 to 13, wherein the cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof.

16. The method of any one of claims 1 to 14, wherein the cell further comprises a reduced amount of CD142 relative to a cell of the same cell type that does not comprise the modification.

17. The method of any one of claims 1 to 15, wherein the cell further comprises reduced expression of CD46 relative to a cell of the same cell type that does not comprise the modification.

18. The method of any one of claims 1 to 16, wherein the cell further comprises reduced expression of CD59 relative to a cell of the same cell type that does not comprise the modification.

19. The method of any one of claims 1 to 17, wherein the cells differentiate from stem cells.

20. The method of claim 18, wherein the stem cells are mesenchymal stem cells.

21. The method of claim 18, wherein the stem cells are embryonic stem cells.

22. The method of claim 18, wherein the stem cell is a pluripotent stem cell, optionally wherein the pluripotent stem cell is an induced pluripotent stem cell.

23. The method of any one of claims 1 to 21, wherein the cells are selected from the group consisting of: cardiac cells, cardiac progenitor cells, neural cells, glial progenitor cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, chimeric Antigen Receptor (CAR) T cells, NK cells, and CAR-NK cells.

24. The method of any one of claims 1 to 22, wherein the cells are derived from primary cells.

25. The method of claim 23, wherein the primary cells are primary T cells, primary beta cells, or primary retinal pigment epithelial cells.

26. The method of claim 24, wherein the cells derived from primary T cells are derived from a T cell pool comprising primary T cells from one or more subjects other than the patient.

27. The method of any one of claims 1 to 25, wherein the cell comprises a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR).

28. The method of claim 26, wherein the antigen binding domain of the CAR binds to CD19, CD22, or BCMA.

29. The method of claim 27, wherein the CAR is a CD 19-specific CAR such that the cell is a CD19 CAR T cell.

30. The method of claim 27, wherein the CAR is a CD 22-specific CAR such that the cell is a CD22 CAR T cell.

31. The method of claim 27, wherein the cell comprises a CD 19-specific CAR and a CD 22-specific CAR, such that the cell is a CD19/CD22 CAR T cell.

32. The method of claim 30, wherein the CD 19-specific CAR and the CD 22-specific CAR are encoded by a single bicistronic polynucleotide.

33. The method of claim 30, wherein the CD 19-specific CAR and the CD 22-specific CAR are encoded by two separate polynucleotides.

34. The method of any one of claims 1 to 32, wherein the first and/or second exogenous polynucleotide is inserted into a genomic locus comprising a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus.

35. The method of claim 33, wherein the first and second genomic loci are the same.

36. The method of claim 33, wherein the first and second genomic loci are different.

37. The method of any one of claims 1 to 35, wherein the cells each further comprise a third exogenous polynucleotide inserted into a third genomic locus.

38. The method of claim 36, wherein the third genomic locus is the same as the first or second genomic locus.

39. The method of claim 36, wherein the third genomic locus is different from the first and/or second genomic loci.

40. The method of any one of claims 33 to 38, wherein the safe harbor locus is selected from the group consisting of: CCR5 gene locus, PPP1R12C (also known as AAVS 1) gene, ROSA26 gene locus and CLYBL gene locus.

41. The method of any one of claims 33 to 38, wherein the target locus is selected from the group consisting of: CXCR4 locus, albumin locus, SHS231 locus, CD142 locus, MICA locus, MICB locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus.

42. The method of claim 39, wherein the insertion into the CCR5 gene locus is at exons 1 to 3, introns 1 to 2, or another coding sequence (CDS) of the CCR5 gene.

43. A method according to claim 39 wherein the insertion into the PPP1R12C gene locus is intron 1 or intron 2 of the PPP1R12C gene.

44. The method of claim 39, wherein the insertion into the CLYBL gene locus is intron 2 of the CLYBL gene.

45. The method of claim 40, wherein the insertion into the ROSA26 gene locus is intron 1 of the ROSA26 gene.

46. The method of claim 40, wherein the insertion into the safe harbor locus is the SHS231 locus.

47. The method of claim 40, wherein the insertion into the CD142 gene locus is at exon 2 of the CD142 gene or another CDs.

48. The method of claim 40 wherein the insertion into the MICA gene locus is at the CDS of the MICA gene.

49. The method of claim 40, wherein the insertion into the MICB gene locus is at the CDS of the MICB gene.

50. The method of any one of claims 33 to 38, wherein the insertion into the B2M gene locus is at exon 2 of the B2M gene or another CDS.

51. The method of any one of claims 33 to 38, wherein the insertion into the CIITA gene locus is at exon 3 of the CIITA gene or another CDS.

52. A method as claimed in any one of claims 33 to 38 wherein the insertion into the TRAC locus is at exon 2 of the TRAC gene or at another CDS.

53. The method of any one of claims 33 to 38, wherein the insertion into the TRB gene locus is at the CDS of the TRB gene.

54. The method of any one of claims 24 to 52, wherein the cells derived from primary T cells comprise one or more of the following that reduce expression:

a. Endogenous T cell receptors;

b. cytotoxic T-lymphocyte-associated protein 4 (CTLA 4);

c. programmed cell death (PD 1); and

d. programmed cell death ligand 1 (PD-L1), wherein the reduced expression is due to a modification and the reduced expression is relative to a cell of the same cell type that does not comprise the modification.

55. The method of claim 53, wherein the cells derived from primary T cells comprise TRAC with reduced expression.

56. The method of any one of claims 22 to 52, wherein the cells are T cells derived from induced pluripotent stem cells comprising one or more of the following for reduced expression:

a. endogenous T cell receptors;

b. cytotoxic T-lymphocyte-associated protein 4 (CTLA 4);

c. programmed cell death (PD 1); and

d. programmed cell death ligand 1 (PD-L1).

57. The method of claim 55, wherein the cells are T cells derived from induced pluripotent stem cells comprising reduced expression TRAC and TRB.

58. The method of any one of claims 1 to 56, wherein the exogenous polynucleotide is operably linked to a promoter.

59. The method of claim 57, wherein the promoter is a CAG and/or EF1a promoter.

60. The method of any one of claims 1 to 58, wherein the population of cells is administered for at least 1 day or more after the patient is sensitive to one or more alloantigens, or for at least 1 day or more after the patient has received the allograft.

61. The method of any one of claims 1 to 58, wherein the population of cells is administered at least one week or more after the patient is sensitive to one or more alloantigens, or at least one week or more after the patient has received the allograft.

62. The method of any one of claims 1-58, wherein the population of cells is administered for at least 1 month or more after the patient is sensitive to one or more alloantigens, and for at least 1 month or more after the patient has received the allograft.

63. The method of any one of claims 1-61, wherein the patient exhibits no immune response after administration of the population of cells.

64. The method of claim 62, wherein the immune-free response after administration of the population of cells is selected from the group consisting of: no systemic immune response, no adaptive immune response, no innate immune response, no T cell response, no B cell response, and no systemic acute cellular immune response.

65. The method of claim 63, wherein the patient exhibits one or more of:

a. after administration of the population of cells, there is no systemic TH1 activation;

b. immune activation of Peripheral Blood Mononuclear Cells (PBMCs) is absent following administration of the population of cells;

c. after administration of the population of cells, no donor-specific IgG antibodies against the population of cells;

d. after administration of the population of cells, no IgM and IgG antibodies are produced against the population of cells; and

e. after administration of the population of cells, there is no cytotoxic T cell killing of the population of cells.

66. The method of any one of claims 1 to 64, wherein the patient is not administered an immunosuppressant for at least 3 days or more before or after administration of the population of cells.

67. The method of any one of claims 1 to 65, wherein the method comprises a dosing regimen comprising:

a. a first administration comprising a therapeutically effective amount of the population of cells;

b. during recovery; and

c. a second administration comprising a therapeutically effective amount of the population of cells.

68. The method of claim 66, wherein said recovery period comprises at least 1 month or more.

69. The method of claim 66, wherein said recovery period comprises at least 2 months or more.

70. The method of any one of claims 66-68, wherein said second administration is initiated when said cells from said first administration are no longer detectable in said patient, optionally wherein said cells are no longer detectable because of removal from a suicide gene or safety switching system.

71. The method of any one of claims 66-69, wherein the low-immunity cells are removed by suicide genes or safety switching systems, and wherein the second administration is initiated when the cells from the first administration are no longer detectable in the patient.

72. The method of any one of claims 66-70, further comprising administering the dosing regimen at least twice.

73. The method of any one of claims 1 to 71, wherein the population of cells is administered for treating a cell defect or as a cell therapy for treating a disorder or disease in a tissue or organ selected from the group consisting of: heart, lung, kidney, liver, pancreas, intestine, stomach, cornea, bone marrow, blood vessels, heart valves, brain, spinal cord, and bone.

74. The method of any one of claims 1 to 72, wherein:

a. The cell deficiency is associated with a neurodegenerative disease or the cell therapy is used to treat a neurodegenerative disease;

b. the cellular defect is associated with a liver disease or the cellular therapy is used to treat a liver disease;

c. the cellular defect is associated with a corneal disease or the cellular therapy is used to treat a corneal disease;

d. the cell deficiency is associated with a cardiovascular disorder or disease or the cell therapy is used to treat a cardiovascular disorder or disease;

e. the cell defect is associated with diabetes or the cell therapy is used to treat diabetes;

f. the cell deficiency is associated with a vascular disorder or disease or the cell therapy is used to treat a vascular disorder or disease;

g. the cell deficiency is associated with autoimmune thyroiditis or the cell therapy is used to treat autoimmune thyroiditis; or (b)

h. The cell deficiency is associated with kidney disease or the cell therapy is used to treat kidney disease.

75. The method of claim 73, wherein:

a. the neurodegenerative disease is selected from the group consisting of: leukodystrophy, huntington's disease, parkinson's disease, multiple sclerosis, transverse myelitis, and Pemetrexed (PMD);

b. The liver disease comprises cirrhosis of the liver;

c. the corneal disease is Fuchs dystrophy or congenital genetic endothelial dystrophy; or (b)

d. The cardiovascular disease is myocardial infarction or congestive heart failure.

76. The method of claim 73 or 74, wherein the population of cells comprises:

a. a cell selected from the group consisting of: glial progenitor cells, oligodendrocytes, astrocytes and dopamine neurons, optionally, wherein the dopamine neurons are selected from the group consisting of: neural stem cells, neural progenitor cells, immature dopamine neurons, and mature dopamine neurons;

b. hepatocytes or hepatic progenitors;

c. corneal endothelial progenitor cells or corneal endothelial cells;

d. cardiomyocytes or cardiac progenitors;

e. pancreatic islet cells, including pancreatic beta islet cells, optionally, wherein the pancreatic islet cells are selected from the group consisting of: pancreatic islet progenitor cells, immature pancreatic islet cells, and mature pancreatic islet cells;

f. endothelial cells;

g. thyroid progenitor cells; or (b)

h. Kidney precursor cells or kidney cells.

77. The method of any one of claims 1 to 75, wherein the population of cells is administered for the treatment of cancer.

78. The method of claim 76, wherein the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

79. The method of any one of claims 1 to 75, wherein the patient is receiving a tissue or organ transplant, optionally wherein the tissue or organ transplant or partial organ transplant is selected from the group consisting of: heart graft, lung graft, kidney graft, liver graft, pancreas graft, intestinal graft, stomach graft, cornea graft, bone marrow graft, vascular graft, heart valve graft, bone graft, partial lung graft, partial kidney graft, partial liver graft, partial pancreas graft, partial intestinal graft, and partial cornea graft.

80. The method of claim 78, wherein the tissue or organ transplant is an allograft transplant.

81. The method of claim 78, wherein the tissue or organ transplant is an autograft transplant.

82. The method of any one of claims 78 to 80, wherein the population of cells is administered for treating a cell defect in a tissue or organ and the tissue or organ transplant is used to replace the same tissue or organ.

83. The method of any one of claims 78 to 80, wherein the population of cells is administered for treating a cellular defect in a tissue or organ and the tissue or organ graft is used to replace a different tissue or organ.

84. The method of any one of claims 78 to 82, wherein the organ transplant is a kidney transplant and the population of cells is a population of pancreatic beta islet cells.

85. The method of claim 83, wherein the patient has diabetes.

86. The method of any one of claims 78 to 82, wherein the organ transplant is a heart transplant and the population of cells is a population of pacing cells.

87. The method of any one of claims 78 to 82, wherein the organ transplant is a pancreatic transplant and the population of cells is a population of beta islet cells.

88. The method of any one of claims 78 to 82, wherein the organ transplant is a partial liver transplant and the population of cells is a population of hepatocytes or hepatic progenitors.

89. Use of a population of low immunity cells for treating a disorder in a patient, wherein the low immunity cells comprise a first exogenous polynucleotide encoding CD47 and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression levels;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient.

90. Use of a population of pancreatic islet cells for treating a disorder in a patient, wherein the pancreatic islet cells comprise a first exogenous polynucleotide encoding CD47 and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient.

91. Use of a population of heart muscle cells for treating a disorder in a patient, wherein the heart muscle cells comprise a first exogenous polynucleotide encoding CD47 and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient.

92. Use of a population of glial progenitor cells for treating a disorder in a patient, wherein the glial progenitor cells comprise a first exogenous polynucleotide encoding CD47 and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient.

93. The use of any one of claims 88 to 91, wherein the patient is a sensitive patient, and wherein the patient exhibits a memory B-cell and/or memory T-cell response to the one or more alloantigens or one or more autoantigens.

94. The use of claim 92, wherein the one or more alloantigens comprise human leukocyte antigens.

95. The use of any one of claims 88 to 93, wherein the patient is a sensitive patient who is sensitive to a previous graft, wherein:

96. The use of any of claims 88 to 93, wherein the patient is a susceptible patient who is susceptible to prior pregnancy, and wherein the patient has previously exhibited an alloimmunity during pregnancy, optionally wherein the alloimmunity during pregnancy is Hemolytic Disease (HDFN) in fetuses and newborns, neonatal Alloimmune Neutropenia (NAN), or fetal and neonatal alloimmune thrombocytopenia (FNAIT).

97. The use of any one of claims 88 to 93, wherein the patient is a susceptible patient who is susceptible to a disease or previous treatment of a disease.

98. The use of any one of claims 88 to 93 or claim 96, wherein the patient receives a prior treatment for a disorder or disease, wherein the prior treatment does not comprise the population of cells, and wherein:

d. the prior treatment is therapeutically effective;

e. the previous treatment is treatment-ineffective;

f. the patient develops an immune response to the prior treatment; and/or

99. The use of claim 97, wherein the prior treatment comprises administering a population of therapeutic cells comprising a suicide gene or a safety switching system, and the immune response occurs in response to activation of the suicide gene or the safety switching system.

100. The use of claim 97, wherein the prior treatment comprises a mechanically assisted treatment, optionally wherein the mechanically assisted treatment comprises hemodialysis or ventricular assist devices.

101. The use of any one of claims 88 to 99, wherein the patient suffers from an allergy, optionally wherein the allergy is an allergy selected from the group consisting of: hay fever, food allergies, insect allergies, drug allergies, and atopic dermatitis.

102. The use of any one of claims 88 to 100, wherein the cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof.

103. The use of any one of claims 88 to 101, wherein the cell further comprises a reduced amount of CD142 relative to a cell of the same cell type that does not comprise the modification.

104. The use of any one of claims 88 to 102, wherein the cell further comprises reduced expression of CD46 relative to a cell of the same cell type that does not comprise the modification.

105. The use of any one of claims 88 to 103, wherein the cell further comprises reduced expression of CD59 relative to a cell of the same cell type that does not comprise the modification.

106. The use of any one of claims 88 to 104, wherein the cells differentiate from stem cells.

107. The use of claim 105, wherein the stem cell is a mesenchymal stem cell.

108. The use of claim 105, wherein the stem cell is an embryonic stem cell.

109. The use of claim 105, wherein the stem cell is a pluripotent stem cell, optionally wherein the pluripotent stem cell is an induced pluripotent stem cell.

110. The use of any one of claims 88 to 108, wherein the cell is selected from the group consisting of: cardiac cells, neural cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigment epithelial cells, hepatocytes, thyroid cells, skin cells, blood cells, plasma cells, platelets, kidney cells, epithelial cells, chimeric Antigen Receptor (CAR) T cells, NK cells, and CAR-NK cells.

111. The use of any one of claims 88 to 109, wherein the cells are derived from primary cells.

112. The use of claim 110, wherein the primary cell is a primary T cell, a primary β cell, or a primary retinal pigment epithelial cell.

113. The use of claim 111, wherein the cells derived from primary T cells are derived from a T cell pool comprising primary T cells from one or more subjects other than the patient.

114. The use of any one of claims 88 to 112, wherein the cell comprises a second exogenous polynucleotide encoding a Chimeric Antigen Receptor (CAR).

115. The use of claim 113, wherein the antigen binding domain of the CAR binds to CD19, CD22 or BCMA.

116. The use of claim 114, wherein the CAR is a CD 19-specific CAR such that the cell is a CD19 CAR T cell.

117. The use of claim 114, wherein the CAR is a CD 22-specific CAR such that the cell is a CD22 CAR T cell.

118. The use of claim 114, wherein the cell comprises a CD 19-specific CAR and a CD 22-specific CAR, such that the cell is a CD19/CD22 CAR T cell.

119. The use of claim 117, wherein the CD 19-specific CAR and the CD 22-specific CAR are encoded by a single bicistronic polynucleotide.

120. The use of claim 117, wherein the CD 19-specific CAR and the CD 22-specific CAR are encoded by two separate polynucleotides.

121. The use of any one of claims 88 to 119, wherein the first and/or second exogenous polynucleotide is inserted into a genomic locus comprising a safe harbor locus, a locus of interest, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus.

122. The use of claim 120, wherein the first and second genomic loci are the same.

123. The use of claim 120, wherein the first and second genomic loci are different.

124. The use of any one of claims 88 to 122, wherein the cells each further comprise a third exogenous polynucleotide inserted into a third genomic locus.

125. The use of claim 123, wherein the third genomic locus is the same as the first or second genomic locus.

126. The use of claim 123, wherein the third genomic locus is different from the first and/or second genomic loci.

127. The use of any one of claims 120 to 125, wherein the safe harbor locus is selected from the group consisting of: CCR5 gene locus, PPP1R12C (also known as AAVS 1) gene and CLYBL gene locus.

128. The use of any one of claims 120 to 125, wherein the locus of interest is selected from the group consisting of: CXCR4 locus, albumin locus, SHS231 locus, ROSA26 locus, CD142 locus, MICA locus, MICB locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

129. The use of claim 126, wherein the insertion into the CCR5 gene locus is at exons 1 to 3, introns 1 to 2 or another coding sequence (CDS) of the CCR5 gene.

130. A use as claimed in claim 126 wherein the insertion into the PPP1R12C gene locus is intron 1 or intron 2 of the PPP1R12C gene.

131. The use of claim 126, wherein the insertion into the CLYBL gene locus is intron 2 of the CLYBL gene.

132. The use of claim 127, wherein the insertion into the ROSA26 gene locus is intron 1 of the ROSA26 gene.

133. The use of claim 127, wherein the insertion into the safe harbor locus is the SHS231 locus.

134. The use of claim 127, wherein the insertion into the CD142 gene locus is at exon 2 of the CD142 gene or another CDs.

135. The use of claim 127, wherein the insertion into the MICA gene locus is at the CDS of the MICA gene.

136. The use of claim 127, wherein the insertion into the MICB gene locus is at the CDS of the MICB gene.

137. The use of any one of claims 120 to 135, wherein the insertion into the B2M gene locus is at exon 2 of the B2M gene or another CDS.

138. The use of any one of claims 120 to 135, wherein the insertion into the CIITA gene locus is at exon 3 of the CIITA gene or another CDS.

139. The use of any one of claims 120 to 135, wherein the insertion into the TRAC locus is at exon 2 of the TRAC gene or at another CDS.

140. The use of any one of claims 120 to 135, wherein the insertion into the TRB gene locus is at the CDS of the TRB gene.

141. The use of any one of claims 111-139, wherein the cells derived from primary T cells comprise one or more of the following that reduce expression:

a. Endogenous T cell receptors;

b. cytotoxic T-lymphocyte-associated protein 4 (CTLA 4);

c. programmed cell death (PD 1); and

142. The use of claim 140, wherein the cells derived from primary T cells comprise reduced expression TRAC.

143. The use of any one of claims 109-139, wherein the cell is a T cell derived from an induced pluripotent stem cell comprising one or more of the following for reduced expression:

a. endogenous T cell receptors;

b. cytotoxic T-lymphocyte-associated protein 4 (CTLA 4);

c. programmed cell death (PD 1); and

144. The use of claim 142, wherein the cell is a T cell derived from an induced pluripotent stem cell comprising reduced expression TRAC and TRB.

145. The use of any one of claims 88 to 143, wherein the exogenous polynucleotide is operably linked to a promoter.

146. The use of claim 144, wherein the promoter is a CAG and/or EF1a promoter.

147. The use of any one of claims 88 to 145, wherein the population of cells is administered for at least 1 day or more after the patient is sensitive to one or more alloantigens, or for at least 1 day or more after the patient has received the allograft.

148. The use of any one of claims 88 to 145, wherein the population of cells is administered at least one week or more after the patient is sensitive to one or more alloantigens, or at least one week or more after the patient has received the allograft.

149. The use of any one of claims 88 to 145, wherein the population of cells is administered for at least 1 month or more after the patient is sensitive to one or more alloantigens, and for at least 1 month or more after the patient has received the allograft.

150. The use of any one of claims 88 to 148, wherein the patient exhibits no immune response after administration of the population of cells.

151. The use of claim 149, wherein the immune-free response after administration of the population of cells is selected from the group consisting of: no systemic immune response, no adaptive immune response, no innate immune response, no T cell response, no B cell response, and no systemic acute cellular immune response.

152. The use of claim 150, wherein the patient exhibits one or more of:

153. The use of any one of claims 88 to 151, wherein the patient has not been administered an immunosuppressant for at least 3 days or more prior to or after administration of the population of cells.

154. The use of any one of claims 88 to 152, wherein the method comprises a dosing regimen comprising:

b. during recovery; and

155. The use of claim 153, wherein the recovery period comprises at least 1 month or more.

156. The use of claim 153, wherein the recovery period comprises at least 2 months or more.

157. The use of any one of claims 153-155, wherein the second administration is initiated when the cells from the first administration are no longer detectable in the patient.

158. The use of any one of claims 153-156, wherein the low-immunity cells are removed by suicide genes or safety switching systems, and wherein the second administration is initiated when the cells from the first administration are no longer detectable in the patient.

159. The use of any one of claims 155-157, further comprising administering the dosing regimen at least twice.

160. The use of any one of claims 88 to 158, wherein the population of cells is administered for treating a cell defect or as a cell therapy for treating a disorder or disease in a tissue or organ selected from the group consisting of: heart, lung, kidney, liver, pancreas, intestine, stomach, cornea, bone marrow, blood vessels, heart valves, brain, spinal cord, and bone.

161. The use of any one of claims 88 to 159, wherein:

162. The use of claim 160, wherein:

b. The liver disease comprises cirrhosis of the liver;

163. The use of claim 160 or 161, wherein the population of cells comprises:

b. hepatocytes or hepatic progenitors;

c. corneal endothelial progenitor cells or corneal endothelial cells;

d. cardiomyocytes or cardiac progenitors;

f. endothelial cells;

g. thyroid progenitor cells; or (b)

h. Kidney precursor cells or kidney cells.

164. The use of any one of claims 88 to 162, wherein the population of cells is administered for the treatment of cancer.

165. The use of claim 163, wherein the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

166. The use of any of claims 88 to 164, wherein the patient is receiving a tissue or organ transplant, optionally wherein the tissue or organ transplant or partial organ transplant is selected from the group consisting of: heart graft, lung graft, kidney graft, liver graft, pancreas graft, intestinal graft, stomach graft, cornea graft, bone marrow graft, vascular graft, heart valve graft, bone graft, partial lung graft, partial kidney graft, partial liver graft, partial pancreas graft, partial intestinal graft, and partial cornea graft.

167. The use of claim 165, wherein the tissue or organ graft is an allograft graft.

168. The use of claim 165, wherein the tissue or organ transplant is an autograft transplant.

169. The use of any one of claims 165-167, wherein the population of cells is administered for treating a cellular defect in a tissue or organ and the tissue or organ transplant is used to replace the same tissue or organ.

170. The use of any of claims 165-168, wherein the population of cells is administered for treating a cellular defect in a tissue or organ and the tissue or organ graft is used to replace a different tissue or organ.

171. The use of any one of claims 165-169, wherein the organ graft is a kidney graft and the population of cells is a population of kidney precursor cells or kidney cells.

172. The use of claim 170, wherein the patient has diabetes.

173. The use of any one of claims 165-169, wherein the organ graft is a heart graft and the population of cells is a population of heart progenitor cells or paced cells.

174. The use of any one of claims 165-169, wherein the organ transplant is a pancreatic transplant and the population of cells is a population of pancreatic beta islet cells.

175. The use of any one of claims 165-169, wherein the organ graft is a partial liver graft and the population of cells is a population of hepatocytes or hepatic progenitors.

176. A method of treating a patient in need thereof, comprising administering a population of low immunity cells, wherein the low immunity cells comprise a first exogenous polynucleotide encoding CD47, a second exogenous polynucleotide encoding a CAR, and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient, wherein the patient:

i. Sensitive to one or more alloantigens;

sensitive to one or more autoantigens;

sensitive due to previous grafts;

sensitivity due to previous pregnancy;

receiving prior treatment for the disorder or disease; and/or

Is a tissue or organ patient and the hypoimmune cells are administered prior to the administration of the tissue or organ transplant.

177. The method of claim 175, wherein the patient is a sensitive patient, and wherein the patient exhibits a memory B-cell and/or memory T-cell response to the one or more alloantigens or one or more autoantigens.

178. The method of claim 176, wherein the one or more alloantigens comprise human leukocyte antigens.

179. The method of any one of claims 175-177, wherein the patient is a sensitive patient that is sensitive to a previous graft, wherein:

180. The method of any one of claims 175-178, wherein the patient is a susceptible patient who is susceptible to prior pregnancy, and wherein the patient has previously exhibited an alloimmunity during pregnancy, optionally wherein the alloimmunity during pregnancy is Hemolytic Disease (HDFN) in the fetus and neonate, neonatal Alloimmune Neutropenia (NAN), or fetal and neonatal alloimmune thrombocytopenia (FNAIT).

181. The method of any one of claims 175-178, wherein the patient is a sensitive patient that is sensitive due to a disorder or prior treatment of a disease.

182. The method of any one of claims 175-178, wherein the patient receives a prior treatment for a disorder or disease, wherein the prior treatment does not comprise the population of cells, and wherein:

d. The prior treatment is therapeutically effective;

e. the previous treatment is treatment-ineffective;

f. the patient develops an immune response to the prior treatment; and/or

183. The method of claim 181, wherein the prior treatment comprises administering a population of therapeutic cells comprising a suicide gene or a safety switching system, and the immune response occurs in response to activation of the suicide gene or the safety switching system.

184. The method of claim 181, wherein the prior therapy comprises a mechanically assisted therapy, optionally wherein the mechanically assisted therapy comprises hemodialysis or ventricular assist devices.

185. The method of any one of claims 1-183, wherein the patient has an allergy, optionally wherein the allergy is an allergy selected from the group consisting of: hay fever, food allergies, insect allergies, drug allergies, and atopic dermatitis.

186. The method of any one of claims 175-184, wherein the cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof.

187. The method of any one of claims 175-185, wherein the cell further comprises reduced expression of CD142 relative to a cell of the same cell type that does not comprise the modification.

188. The method of any one of claims 175-186, wherein the cell further comprises reduced expression of CD46 relative to a cell of the same cell type that does not comprise the modification.

189. The method of any one of claims 175-187, wherein the cell further comprises reduced expression amounts of CD59 relative to a cell of the same cell type that does not comprise the modification.

190. The method of any one of claims 175-188, wherein the cells differentiate from stem cells.

191. The method of claim 189, wherein the stem cells are mesenchymal stem cells.

192. The method of claim 189, wherein the stem cells are embryonic stem cells.

193. The method of claim 189, wherein the stem cell is a pluripotent stem cell, optionally wherein the pluripotent stem cell is an induced pluripotent stem cell.

194. The method of any one of claims 175-192, wherein the cell is a CAR T cell or a CAR-NK cell.

195. The method of any one of claims 175-193, wherein the cells are derived from primary T cells.

196. The method of claim 194, wherein the cells are derived from a T cell pool comprising primary T cells from one or more subjects other than the patient.

197. The method of any one of claims 175-195, wherein the antigen binding domain of the CAR binds to CD19, CD22, or BCMA.

198. The method of claim 196, wherein the CAR is a CD 19-specific CAR such that the cell is a CD19 CAR T cell.

199. The method of claim 196, wherein the CAR is a CD 22-specific CAR such that the cell is a CD22 CAR T cell.

200. The method of claim 196, wherein the cell comprises a CD 19-specific CAR and a CD 22-specific CAR, such that the cell is a CD19/CD22 CAR T cell.

201. The method of claim 199, wherein the CD 19-specific CAR and the CD 22-specific CAR are encoded by a single bicistronic polynucleotide.

202. The method of claim 199, wherein the CD 19-specific CAR and the CD 22-specific CAR are encoded by two separate polynucleotides.

203. The method of any one of claims 175 to 201, wherein the first and/or second exogenous polynucleotide is inserted into a genomic locus comprising a safe harbor locus, a target locus, a B2M locus, a CIITA locus, a TRAC locus, or a TRB locus.

204. The method of claim 202, wherein the first and second genomic loci are the same.

205. The method of claim 202, wherein the first and second genomic loci are different.

206. The method of any one of claims 175-204, wherein the cells each further comprise a third exogenous polynucleotide inserted into a third genomic locus.

207. The method of claim 206, wherein the third genomic locus is the same as the first or second genomic locus.

208. The method of claim 206, wherein the third genomic locus is different from the first and/or second genomic loci.

209. The method of any one of claims 202 to 207, wherein the safe harbor locus is selected from the group consisting of: CCR5 gene locus, PPP1R12C (also known as AAVS 1) gene and CLYBL gene locus.

210. The method of any one of claims 202-207, wherein the target locus is selected from the group consisting of: CXCR4 locus, albumin locus, SHS231 locus, ROSA26 locus, CD142 locus, MICA locus, MICB locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

211. The method of claim 208, wherein the insertion into the CCR5 gene locus is at exons 1 to 3, introns 1 to 2, or another coding sequence (CDS) of the CCR5 gene.

212. A method as in claim 208 wherein the insertion into the PPP1R12C gene locus is intron 1 or intron 2 of the PPP1R12C gene.

213. The method of claim 208 wherein the insertion into the CLYBL gene locus is intron 2 of the CLYBL gene.

214. The method of claim 209, wherein the insertion into the ROSA26 gene locus is intron 1 of the ROSA26 gene.

215. The method of claim 209, wherein the insertion into the safe harbor locus is the SHS231 locus.

216. The method of claim 209, wherein the insertion into the CD142 gene locus is at exon 2 of the CD142 gene or another CDs.

217. The method of claim 209, wherein the insertion into the MICA gene locus is at the CDS of the MICA gene.

218. The method of claim 209, wherein the insertion into the MICB gene locus is at the CDS of the MICB gene.

219. The method of any one of claims 202-217, wherein the insertion into the B2M gene locus is at exon 2 of the B2M gene or another CDS.

220. The method of any one of claims 202-217, wherein the insertion into the CIITA gene locus is at exon 3 of the CIITA gene or another CDS.

221. The method of any one of claims 202-217, wherein the insertion into the TRAC locus is at exon 2 of the TRAC gene or at another CDS.

222. The method of any one of claims 202-217, wherein the insertion into the TRB gene locus is at the CDS of the TRB gene.

223. The method of any one of claims 194-221, wherein the cells derived from primary T cells comprise one or more of the following that reduce expression:

a. Endogenous T cell receptors;

b. cytotoxic T-lymphocyte-associated protein 4 (CTLA 4);

c. programmed cell death (PD 1); and

224. The method of claim 222, wherein the cells derived from primary T cells comprise reduced expression TRAC.

225. The method of any one of claims 193 to 221, wherein the cell is a T cell derived from an induced pluripotent stem cell comprising one or more of the following for reduced expression:

a. endogenous T cell receptors;

b. cytotoxic T-lymphocyte-associated protein 4 (CTLA 4);

c. programmed cell death (PD 1); and

226. The method of claim 224, wherein the cells are T cells derived from induced pluripotent stem cells comprising reduced expression TRAC and TRB.

227. The method of any one of claims 175-225, wherein the exogenous polynucleotide is operably linked to a promoter.

228. The method of claim 226, wherein the promoter is a CAG and/or EF1a promoter.

229. The method of any one of claims 175-227, wherein the population of cells is administered for at least 1 day or more after the patient is sensitive to one or more alloantigens, or for at least 1 day or more after the patient has received the allograft.

230. The method of any one of claims 175-227, wherein the population of cells is administered at least one week or more after the patient is sensitive to one or more alloantigens, or at least one week or more after the patient has received the allograft.

231. The method of any one of claims 175-227, wherein the population of cells is administered for at least 1 month or more after the patient is sensitive to one or more alloantigens, and for at least 1 month or more after the patient has received the allograft.

232. The method of any one of claims 175-230, wherein the patient exhibits no immune response after administration of the population of cells.

233. The method of claim 231, wherein the immune-free response after administration of the population of cells is selected from the group consisting of: no systemic immune response, no adaptive immune response, no innate immune response, no T cell response, no B cell response, and no systemic acute cellular immune response.

234. The method of claim 232, wherein the patient exhibits one or more of:

235. The method of any one of claims 175-233, wherein the patient has not been administered an immunosuppressant for at least 3 days or more prior to or after administration of the population of cells.

236. The method of any one of claims 175-234, wherein the method comprises a dosing regimen comprising:

b. during recovery; and

237. The method of claim 235, wherein the recovery period comprises at least 1 month or more.

238. The method of claim 235, wherein the recovery period comprises at least 2 months or more.

239. The method of any one of claims 235-237, wherein the second administration is initiated when the cells from the first administration are no longer detectable in the patient.

240. The method of any one of claims 235-238, wherein the low-immunity cells are removed by suicide genes or safety switching systems, and wherein the second administration is initiated when the cells from the first administration are no longer detectable in the patient.

241. The method of any one of claims 235-239, further comprising administering the dosing regimen at least twice.

242. The method of any one of claims 175-240, wherein the population of cells is administered for treating cancer.

243. The method of claim 241, wherein the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

244. Use of a population of low immunity cells for treating a disorder in a patient, wherein the low immunity cells comprise a first exogenous polynucleotide encoding CD47, a second exogenous polynucleotide encoding a CAR, and

(I) One or more of the following:

b. reduced expression of MHC class I and class II human leukocyte antigens;

d. B2M and CIITA with reduced expression;

(II) wherein:

a. the patient is not a sensitive patient; or (b)

b. The patient is a sensitive patient.

245. The use of claim 243, wherein said patient is a sensitive patient, and wherein said patient exhibits a memory B-cell and/or memory T-cell response to said one or more alloantigens or one or more autoantigens.

246. The use of claim 244, wherein the one or more alloantigens comprise human leukocyte antigens.

247. The use of any one of claims 243-245, wherein said patient is a sensitive patient who is sensitive to a previous graft, wherein:

248. The use of any one of claims 243 to 245, wherein said patient is a susceptible patient who is susceptible to prior pregnancy, and wherein said patient has previously exhibited an alloimmunity during pregnancy, optionally wherein said alloimmunity during pregnancy is Hemolytic Disease (HDFN) in the fetus and neonate, neonatal Alloimmune Neutropenia (NAN) or fetal and neonatal alloimmune thrombocytopenia (FNAIT).

249. The use of any one of claims 243-245, wherein said patient is a susceptible patient susceptible to a disorder or prior treatment of a disease.

250. The use of any one of claims 243-245, wherein said patient received a prior treatment for a disorder or disease, wherein said prior treatment did not comprise said population of cells, and wherein:

c. the population of cells exhibits a longer therapeutic effect on the treatment of the disorder or disease in the patient than the prior treatment; the prior treatment is therapeutically effective;

d. the previous treatment is treatment-ineffective;

e. the patient develops an immune response to the prior treatment; and/or

f. The population of cells is administered for the treatment of a condition or disease that is different from the previous treatment.

251. The use of claim 249, wherein the prior treatment comprises administration of a population of therapeutic cells comprising a suicide gene or a safety switching system, and the immune response occurs in response to activation of the suicide gene or the safety switching system.

252. The use of claim 249, wherein the prior treatment comprises a mechanically assisted treatment, optionally wherein the mechanically assisted treatment comprises hemodialysis or ventricular assist devices.

253. The use of any one of claims 243-251, wherein said patient has an allergy, optionally wherein said allergy is an allergy selected from the group consisting of: hay fever, food allergies, insect allergies, drug allergies, and atopic dermatitis.

254. The use of any one of claims 243 to 252, wherein said cell further comprises one or more exogenous polypeptides selected from the group consisting of: DUX4, CD24, CD46, CD55, CD59, CD200, PD-L1, HLA-E, HLA-G, IDO, fasL, IL-35, IL-39, CCL21, CCL22, mfge8, serpin B9, and combinations thereof.

255. The use of any one of claims 243 to 253, wherein said cell further comprises a reduced amount of CD142 relative to a cell of the same cell type that does not comprise a modification.

256. The use of any one of claims 243 to 254, wherein said cell further comprises a reduced amount of CD46 relative to a cell of the same cell type that does not comprise a modification.

257. The use of any one of claims 243 to 255, wherein said cell further comprises a reduced amount of CD59 relative to a cell of the same cell type that does not comprise a modification.

258. The use of any one of claims 243 to 256, wherein the cell is differentiated from a stem cell.

259. The use of claim 257, wherein the stem cells are mesenchymal stem cells.

260. The use of claim 257, wherein the stem cells are embryonic stem cells.

261. The use of claim 257, wherein the stem cells are pluripotent stem cells, optionally wherein the pluripotent stem cells are induced pluripotent stem cells.

262. The use of any one of claims 243 to 260, wherein said cell is a CAR T cell or a CAR-NK cell.

263. The use of any one of claims 243 to 261, wherein said cells are derived from primary T cells.

264. The use of claim 262, wherein the cell is derived from a T cell pool comprising primary T cells from one or more subjects other than the patient.

265. The use of any one of claims 243 to 263, wherein the antigen binding domain of the CAR binds to CD19, CD22, or BCMA.

266. The use of claim 264, wherein the CAR is a CD 19-specific CAR, such that the cell is a CD19 CAR T cell.

267. The use of claim 264, wherein the CAR is a CD 22-specific CAR, such that the cell is a CD22 CAR T cell.

268. The use of claim 264, wherein the cell comprises a CD 19-specific CAR and a CD 22-specific CAR, such that the cell is a CD19/CD22 CAR T cell.

269. The use of claim 267, wherein the CD 19-specific CAR and the CD 22-specific CAR are encoded by a single bicistronic polynucleotide.

270. The use of claim 267, wherein the CD 19-specific CAR and the CD 22-specific CAR are encoded by two separate polynucleotides.

271. The use of any one of claims 243 to 269, wherein said first and/or second exogenous polynucleotide is inserted into a genomic locus comprising a safe harbor locus, a locus of interest, a B2M locus, a CIITA locus, a TRAC locus or a TRB locus.

272. The use of claim 270, wherein the first and second genomic loci are the same.

273. The use of claim 270, wherein the first and second genomic loci are different.

274. The use of any one of claims 243 to 272, wherein said cells each further comprise a third exogenous polynucleotide inserted into a third genomic locus.

275. The use of claim 273, wherein the third genomic locus is the same as the first or second genomic locus.

276. The use of claim 273, wherein the third genomic locus is different from the first and/or second genomic loci.

277. The use of any one of claims 270 to 275, wherein the safe harbor locus is selected from the group consisting of: CCR5 gene locus, PPP1R12C (also known as AAVS 1) gene and CLYBL gene locus.

278. The use of any one of claims 270 to 275, wherein the target locus is selected from the group consisting of: CXCR4 locus, albumin locus, SHS231 locus, ROSA26 locus, CD142 locus, MICA locus, MICB locus, LRP1 locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus and KDM5D locus.

279. The use of claim 276, wherein the insertion into the CCR5 gene locus is at exons 1 to 3, introns 1 to 2, or another coding sequence (CDS) of the CCR5 gene.

280. The use of claim 276, wherein the insertion into the PPP1R12C gene locus is intron 1 or intron 2 of the PPP1R12C gene.

281. The use of claim 276, wherein the insertion into the CLYBL gene locus is intron 2 of the CLYBL gene.

282. The use of claim 277, wherein the insertion into the ROSA26 gene locus is intron 1 of the ROSA26 gene.

283. The use of claim 277, wherein the insertion into the safe harbor locus is the SHS231 locus.

284. The use of claim 277, wherein the insertion into the CD142 gene locus is at exon 2 of the CD142 gene or another CDs.

285. The use of claim 277, wherein the insertion into the MICA gene locus is at the CDS of the MICA gene.

286. The use of claim 277, wherein the insertion into the MICB gene locus is at the CDS of the MICB gene.

287. The use of any one of claims 270 to 285, wherein the insertion into the B2M gene locus is at exon 2 of the B2M gene or another CDS.

288. The use of any one of claims 270 to 285, wherein the insertion into the CIITA gene locus is at exon 3 of the CIITA gene or another CDS.

289. The use of any one of claims 270 to 285, wherein the insertion into the TRAC locus is at exon 2 of the TRAC gene or at another CDS.

290. The use of any one of claims 270 to 285, wherein the insertion into the TRB gene locus is at the CDS of the TRB gene.

291. The use of any one of claims 262-289, wherein the cells derived from primary T cells comprise one or more of the following that reduce expression:

a. endogenous T cell receptors;

b. cytotoxic T-lymphocyte-associated protein 4 (CTLA 4);

c. programmed cell death (PD 1); and

292. The use of claim 290, wherein the cells derived from primary T cells comprise reduced expression TRAC.

293. The use of any one of claims 261-289, wherein the cell is a T cell derived from an induced pluripotent stem cell comprising one or more of the following for reduced expression:

a. endogenous T cell receptors;

b. cytotoxic T-lymphocyte-associated protein 4 (CTLA 4);

c. programmed cell death (PD 1); and

294. The use of claim 292, wherein the cell is a T cell derived from an induced pluripotent stem cell comprising reduced expression TRAC and TRB.

295. The use of any one of claims 243 to 293, wherein said exogenous polynucleotide is operably linked to a promoter.

296. The use of claim 294, wherein the promoter is a CAG and/or EF1a promoter.

297. The use of any one of claims 243-295, wherein said population of cells is administered for at least 1 day or more after said patient is sensitive to one or more alloantigens, or for at least 1 day or more after said patient has received said allograft.

298. The use of any one of claims 243-295, wherein said population of cells is administered for at least one week or more after said patient is sensitive to one or more alloantigens, or for at least one week or more after said patient has received said allograft.

299. The use of any one of claims 243-295, wherein said population of cells is administered for at least 1 month or more after said patient is susceptible to one or more alloantigens, and for at least 1 month or more after said patient has received said allograft.

300. The use of any one of claims 243-298, wherein, after administration of said population of cells, said patient exhibits no immune response.

301. The use of claim 299, wherein the immune-free response after administration of the population of cells is selected from the group consisting of: no systemic immune response, no adaptive immune response, no innate immune response, no T cell response, no B cell response, and no systemic acute cellular immune response.

302. The use of claim 300, wherein the patient exhibits one or more of:

303. The use of any one of claims 243 to 301, wherein said patient has not been administered an immunosuppressant for at least 3 days or more prior to or after administration of said population of cells.

304. The use of any one of claims 243 to 302, wherein said method comprises a dosing regimen comprising:

b. during recovery; and

305. The use of claim 303, wherein the recovery period comprises at least 1 month or more.

306. The use of claim 303, wherein the recovery period comprises at least 2 months or more.

307. The use of any one of claims 303-305, wherein the second administration is initiated when the cells from the first administration are no longer detectable in the patient.

308. The use of any one of claims 303 to 306, wherein the low immunity cells are removed by a suicide gene or safety switching system, and wherein the second administration is initiated when the cells from the first administration are no longer detectable in the patient.

309. The use of any one of claims 303-307, further comprising administering the dosing regimen at least twice.

310. The use of any one of claims 243-308, wherein said population of cells is administered for the treatment of cancer.

311. The use of claim 309, wherein the cancer is selected from the group consisting of: b-cell acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma, liver cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, lung cancer, non-small cell lung cancer, acute myelogenous leukemia, multiple myeloma, gastric cancer, gastric adenocarcinoma, pancreatic adenocarcinoma, glioblastoma, neuroblastoma, lung squamous cell carcinoma, hepatocellular carcinoma, and bladder cancer.

312. The use of claim 97 or 249 or the method of claim 181, wherein the prior treatment comprises an allogeneic CAR-T cell-based therapy or an autologous CAR-T cell-based therapy, wherein the autologous CAR-T cell-based therapy is selected from the group consisting of: bucarba Ji Aolun, sicalico, ai Kaba Ji Weisai, li Jimai Racemosaicism, te Sha Jinlu, descartes-08 or Descartes-11 from Cartesian Therapeutics, CTL110 from Novartis, P-BMCA-101 from Poseida Therapeutics, AUTO4 from Autolus Limited, UCARTCS from Cellectis, PBCAR19B or PBCAR269A from Precision Biosciences, FT819 from Fate Therapeutics and CYAD-211 from Clyad Oncology.