Comprehensively immune protected cells

Abstract

The invention provides therapeutic cells that are comprehensively immune protected having a reduced or eliminated surface leukocyte antigen class I, an enhanced SIRPa engager (SIRPaE) expression, and an enhanced Fc sequestration molecule expression. In some aspects, the cells of the invention engage one or both of CD200R or CD99 ligands on immune cells. In other aspects, the cells of the invention sequester antibodies to inhibit humoral immunity. In other aspects, the cells of the invention have reduced or eliminated leukocyte antigens from both class I and class II. In other embodiments, the cells of the invention are ABO or Rh compatible with a recipient subject. In other embodiments, the cells of the invention are allogeneic or xenogeneic to the recipient.

Description

RUC027WSF2024-223COMPREHENSIVELY IMMUNE PROTECTED CELLSI. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 699,633, filed September 26, 2024, and U.S. Provisional Application No. 63 / 762,505, filed February 24, 2025, and are incorporated herein by reference in their entirety.II. FIELD OF THE INVENTION

[0002] The invention provides therapeutic cells that are comprehensively immune protected having a reduced or eliminated surface leukocyte antigen class I, an enhanced SIRPa engager (SIRPaE) expression, and an enhanced Fc sequestration molecule expression. Thus, CIP cells are protected from allogeneic adaptive immune cells, allogeneic innate immune cells, and antibody-mediated killing. In some aspects, the cells of the invention engage one or both of CD200R or CD99 ligands (e.g. PILRa and CD99) on immune cells. In other aspects, the cells of the invention sequester antibodies to inhibit humoral immunity. In other aspects, the cells of the invention have reduced or eliminated leukocyte antigens from both class I and class II. In other embodiments, the cells of the invention are ABO or Rh compatible with a recipient subject. In other embodiments, the cells of the invention are allogeneic or xenogeneic to the recipient. These comprehensively immune protected cells have a reduced susceptibility to the recipient's immune system that would otherwise kill them following transplantation. In some embodiments, the therapeutic cells are used in a regenerative cell therapy for treating injured organs, tissues, or to replace functional cells orthotopically or heterotopically. In other embodiments, the cell therapies of the invention comprise chimeric antigen receptor (CAR) cells, immune cells expressing an endogenous or exogenous T cell receptor (TCR), immune cells, endothelial cells, fibrocytes, dopaminergic neurons, pancreatic islet cells, hepatocytes, cardiomyocytes, retinal pigment endothelium cells, thyroid cells, or parathyroid cells that are used for treating diseases or rehabilitating damaged tissues.III. BACKGROUND OF THE INVENTION

[0003] Regenerative cell therapy is an important potential treatment for regenerating injured organs and tissue. With the low availability of organs for transplantation and the accompanying lengthy wait, the possibility of regenerating tissue by transplanting readily available cell lines into patients is understandably appealing. Regenerative cell therapy hasRUC027WSF2024-223 shown promising initial results for rehabilitating damaged tissues after transplantation in animal models (e.g. after myocardial infarction). The propensity for the transplant recipient's immune system to reject allogeneic or xenogeneic material, however, greatly reduces the potential efficacy of therapeutics and diminishes the possible positive effects surrounding such treatments.

[0004] Autologous induced pluripotent stem cells (iPSCs) theoretically constitute an unlimited cell source for patient-specific cell-based organ repair strategies. Their generation, however, poses technical and manufacturing challenges and is a lengthy process that conceptually prevents any acute treatment modalities. Allogeneic iPSC-based therapies or embryonic stem cell-based therapies are easier from a manufacturing standpoint and allow the generation of well-screened, standardized, high-quality cell products. Because of their allogeneic origin, however, such cell products would undergo rejection. With the reduction or elimination of the cells’ antigenicity, universally-acceptable cell products could be produced. Because pluripotent stem cells can be differentiated into any cell type of the three germ layers, the potential application of stem cell therapy is wide-ranging. Differentiation can be performed ex vivo or in vivo by transplanting progenitor cells that continue to differentiate and mature in the organ environment of the implantation site. Ex vivo differentiation allows researchers or clinicians to closely monitor the procedure and ensures that the proper population of cells is generated prior to transplantation.

[0005] Organ transplantation is the ideal treatment for most end-stage organ failure affecting the heart, lungs, kidneys, liver, and in certain cases, the pancreas or other endocrine organs like the thyroid or parathyroid glands. The demand for human organs for transplantation purpose, however, far exceeds the number of organ donations. This limits this procedure in general clinical practice. The World Health Organization (Geneva) estimates that only 10% of the worldwide need for organ transplantation is being met. Currently, ten patients die each day in the United States while waiting for lifesaving vital organ transplants. Moreover, transplantation of cells and tissues may be therapeutic for certain diseases such as neurodegenerative disorders and endocrine diseases like diabetes, hypothyroidism or hypoparathyroidi sm .

[0006] Xenotransplantation is any procedure that involves the transplantation, implantation or infusion into a human recipient of either (a) live cells, tissues, or organs from a nonhuman animal source, or (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs. Immune rejection remains the majorRUC027WSF2024-223 challenge to overcome in xenotransplantation. There are three different types of xenogeneic rejections: hyperacute rejection (HAR), delayed xenograft rejection (DXR) and chronic rejection. DXR sometimes involves acute humoral xenograft rejection (AHR) and cellular xenograft rejection (CXR). These cannot be strictly distinguished from each other pathologicaly.

[0007] Rhesus monkeys are important immunological models for interventional new drug (IND)-enabling studies. Human cell products will need to undergo non-human primate studies before entering clinical trials. There are, however, additional xenogeneic immune barriers that induce monkey PMNs to kill transplanted human cells. Likewise, there are xenogeneic immune barriers that induce human PMNs to kill transplanted animal cells and organs. Examples include transplanted pig cells, tissues, and organs.

[0008] During evolution, the enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH) has been inactivated in a number of lineages, including humans. CMAH is responsible for the synthesis of N-glycolylneuraminic acid (Neu5Gc), a sialic acid that is present in most mammals but not in humans. It is found in the tips of glycolipids, glycoproteins, and carbohydrate chains. Instead, humans express the sialic acids N-acetylneuraminic acid (Neu5Ac).

[0009] The art seeks cells or stem cells capable of producing such cells that are used to regenerate or replace diseased or deficient cells, tissues, or organs. Pluripotent stem cells (PSCs) may be used as a source for producing differentiated cell products because they rapidly propagate and differentiate into many possible cell types. The family of PSCs includes several members generated via different techniques and possessing distinct immunogenic features. Patient compatibility with engineered cells or tissues derived from PSCs determines the risk of immune rejection and the requirement for immunosuppression co-therapies.

[0010] Embryonic stem cells (ESCs) isolated from the inner cell mass of blastocysts exhibit histocompatibility antigens that are mismatches with virtually all possible recipients. This immunological barrier cannot be solved by human leukocyte antigen (HLA)-typed banks of ESCs because even HLA-matched PSC grafts undergo rejection because of mismatches in non-HLA molecules that function as minor antigens. This is also true for allogeneic induced pluripotent stem cells (iPSCs).RUC027WSF2024-223

[0011] Hypoimmune or hypoimmunogenic cells (z.e.hypoimmune cells), pluripotent (HIP) cells, and HIP cell derivatives have gene knockouts or transgenes to protect them from the cellular components of the immune system that include T cells, NK cells, and macrophages. They may also be ABO blood group type O and Rh negative (HIPO-).

[0012] Transplanted cells, tissues, or organs may be killed via antibody-dependent cellular cytotoxicity (ADCC) or by NK cells, macrophages, B-cells, or granulocytes. Additionally, they may be killed via complement-dependent cytotoxicity (CDC) through the activation of the complement cascade. All of those killing mechanisms utilize antibodies that bind to a target cell and activate the effector immune cells or complement.

[0013] Polymorphonuclear cells (PMNs) are a category of white blood cells characterized by the presence of granules in their cytoplasm and lobed nuclei that are usually in three segments. This distinguishes them from the mononuclear immune cell fraction that contains lymphocytes, macrophages, and NK cells. Neutrophils are the most abundant of the PMNs. Eosinophils, basophils, and mast cells are less frequent PMNs. They are found in the bloodstream or particular tissues. PMNs are the most abundant type of phagocytes, constituting 60% to 65% of the total circulating white blood cells. Once PMNs have received activation signals, it takes them about thirty minutes to leave the blood and reach the site of infection or tissue damage.

[0014] PMNs do not return to the blood. They exert their effector responses and die. PMNs have three mechanisms to attack, contain, and kill evading micro-organisms: 1) Phagocytosis. PMNs are professional phagocytes and rapidly engulf invaders coated with antibodies and complement as well as damaged cells or cellular debris. 2) PMNs secrete cytotoxic granules containing reactive oxygen species, cationic proteins and defensins, lysozyme, myeloperoxidase, proteolytic enzymes and cathepsin G. 3) Formation of neutrophil extracellular traps (NETs). NETs comprise a web of chromatin and serine proteases that trap and kill microbes extracellularly.

[0015] PMNs are often the first line of defense. They quickly migrate to sites of injury and infection. Because their defense mechanisms are very unspecific, they may cause significant collateral damage to healthy cells in the vicinity. They are attracted and activated by cytokines, chemokines, pathogen-associated molecular patterns (PAMPs) and danger- associated molecular patterns (DAMPs). The latter activate pattern recognition receptors (PRRs) including toll-like receptors (TLRs). PMNs overall have multiple activating or inhibitory receptors.RUC027WSF2024-223

[0016] Thus, transplanted cell death not only reduces the efficacy of the cell product but also triggers or amplifies innate defense mechanisms involving PMNs. PMN responses localized to the time and location of transplantation that contain and scavenge debris can also kill living cells in the vicinity as collateral damage. This further reduces cell survival and intensifies the PMN response. Strategies to prevent relevant PMN activation will facilitate cell transplantation and improve the potency of cell products.IV. SUMMARY OF THE INVENTION

[0017] The invention provides therapeutic cells that are comprehensively immune protected. A comprehensively immune protected (CIP) cell comprises a reduced or eliminated surface leukocyte antigen class I, a SIRPa engager (SIRPaE), and an Fc sequestration molecule. Thus, CIP cells are protected from allogeneic adaptive immune cells, allogeneic innate immune cells, and antibody-mediated killing. In some aspects, the cells of the invention engage one or both of CD200R or CD99 ligands (e.g. PILRa and CD99) for xenogeneic transplantation.

[0018] Thus, the invention provides a cell, comprising a reduced or eliminated surface leukocyte antigen class I, a first cell-surface molecule that is a SIRPa engager molecule (SIRPaE) expressed on a cell surface membrane of the cell by a first transmembrane domain, wherein the first cell-surface molecule does not comprise a domain that induces intracellular signaling, and a second cell-surface molecule that sequesters antibodies expressed on the cell surface membrane comprising a CD64 extracellular domain bound to the cell surface by a second transmembrane domain. In some aspects, either the first or the second transmembrane domain, or both, comprise at least a 90% sequence identity to SEQ ID NO:6. In other aspects, either the first or the second transmembrane domain, or both, comprise the sequence of SEQ ID N0:6.

[0019] The invention provides cells as disclosed herein, wherein the SIRPaE has a CD47 extra-cellular domain having at least a 90% sequence identity to SEQ ID NO:44 and a transmembrane domain having at least a 90% sequence identity to SEQ ID NO:6. In some aspects, the CD47 extra-cellular domain has the sequence of SEQ ID NO:44 and the transmembrane domain has the sequence of SEQ ID NO:6.

[0020] In some aspects of the invention, the SIRPaE has at least a 90% sequence identity to SEQ ID NO:27. In other aspects, the SIRPaE has the sequence of SEQ ID NO:27. In otherRUC027WSF2024-223 aspects, the SIRPaE has at least a 90% sequence identity to any one of SEQ ID NOS:23, 38, 39, 40, or 41. In other aspects, the SIRPaE has the sequence of any one of SEQ ID NOS:23, 38, 39, 40, or 41.

[0021] The invention provides cells as disclosed herein, wherein the CD64 extracellular domain has at least a 90% sequence identity to SEQ ID NO:45. In other aspects, the CD64 extracellular domain has the sequence of SEQ ID NO:45. In other aspects, the antibody sequestration molecule has CD64 activity and has at least a 90% sequence identity to SEQ ID NO:24. In other aspects, the antibody sequestration molecule has the sequence of SEQ ID NO:24. In other aspects, the antibody sequestration molecule has CD64 activity and has at least a 90% sequence identity to SEQ ID NO: 14. In other aspects, the antibody sequestration molecule has the sequence of SEQ ID NO: 14.

[0022] The invention provides cells as disclosed herein, further comprising a third cell surface molecule, wherein the third cell-surface molecule is a CD200R engager molecule, wherein the CD200R engager molecule has a CD200R engaging domain that engages a CD200R on immune cells following transplantation of the cell into a subject. In some aspects, the third cell surface molecule is a chimera. In other aspects, the CD200R engaging domain comprises an immunoglobulin heavy chain domain. In other aspects, the third cell surface molecule lacks a CD200 intracellular signaling function. In preferred aspects, the immunoglobulin heavy chain domain is a variable heavy chain domain having at least a 90% sequence identity to SEQ ID NO: 1. In other preferred aspects, the variable heavy chain domain comprises the sequence of SEQ ID NO: 1.

[0023] In other aspects of the invention, the third cell surface molecule further comprises an immunoglobulin light chain domain. In other aspects, the immunoglobulin light chain domain has at least a 90% sequence identity to SEQ ID NO:2. In other aspects, the immunoglobulin light chain domain comprises the sequence of SEQ ID NO:2. In other aspects, the third cell surface molecule is a protein monomer, dimer, or multimer that comprises the amino acid sequences of both SEQ ID NO: 1 and SEQ ID NO:2. In other aspects, the third cell surface molecule has at least a 90% sequence identity to SEQ ID NO:7. In other aspects, the third cell surface molecule has the sequence of SEQ ID NO:7.

[0024] In other aspects of the invention, the third cell surface molecule comprises an antibody Fab or a single chain variable fragment (scFV) that binds to CD200R. In other aspects, the Fab or scFV binds to CD200R with an affinity measured by its dissociation constant (Kd), wherein the Kd is between about 107and 1013M.RUC027WSF2024-223

[0025] In other aspects of the invention, the third cell surface molecule is a fusion protein comprising a heterologous transmembrane domain (TMD). In other aspects, the TMD comprises a single a helix, multiple a helices, or a rolled-up P sheet. In other aspects, the TMD has at least a 90% sequence identity to SEQ ID NO:6. In other aspects, the TMD has the sequence of SEQ ID NO:6.

[0026] In some aspects of the invention, the TMD is selected from the group consisting of CD99, CD47, CD64, CD85f, CD349, CD284, CD261, CD 172b, CD277, CD 186, CD 156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CDla, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CDl lb, CD107a, CD331, CD222, CD20, CD79a, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD 119, CD 19, CD 116, CD46, CDle, CD Id, CD227, CD44, CD62P, CD 104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CDl la, CDl lc, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CDlb, CDlc, CD154, CD6, CD52, CD132, CD32, CD66b, CD171, CD191, CD197, CD185, CD131, CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361, CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231, CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD158i, CD178, CD295, CD151, CD97, CD183, CD39, CD239, CD193, CD194, CD195, CD196, CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241, CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91, CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161, CD215, CD150, CD l id, CD218a, CD210, CD 166, CD 162, CD213a2, CD242, CD 158g, CD158h, CD279, CD111, CD281, CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158z, CD158f2, CD85j, CD300f, CD92, CD351, CD112, CD100, CD270, CD101, CD297, CD316, CD352,RUC027WSF2024-223CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD 114, CD 180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201, CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51, CD41, CD29, CD 18, CD61, and CD 104.

[0027] The cell of claim 34, wherein the third cell surface molecule has at least a 90% sequence identity to SEQ ID NO:28. In other aspects, the third cell surface molecule has the sequence of SEQ ID NO:28.

[0028] In other aspects of the invention, the third cell surface molecule does not have an intracellular domain (ICD) or does not transmit an intracellular signal. In other aspects, the third cell surface molecule further comprises a cytoplasmic domain that does not transmit an intracellular signal. In other aspects, the third cell surface molecule has an intracellular domain from CD16, CD32, CD64, CD8, CD3, CD28, CD137, SEQ ID NO:42, or SEQ ID NO:43.

[0029] In some aspects of the invention, the third cell surface molecule has CD200 activity and has at least a 90% sequence identity to SEQ ID NO:22. In other apsects, the third cell surface molecule has the sequence of SEQ ID NO:22. In other aspects, the third cell surface molecule has CD200 activity and comprises a CD200 extracellular domain with at least a 90% sequence identity to SEQ ID NO:29. In other aspects, the third cell surface molecule has CD200 activity and comprises a CD200 extracellular domain having the sequence of SEQ ID NO:29.

[0030] The invention provides cells as disclosed herein, further comprising a fourth cellsurface molecule that engages CD99 ligands. In some aspects, the fourth cell-surface molecule has a CD99 extra-cellular function and has at least a 90% sequence identity to SEQ ID NO:21. In other aspects, the fourth cell-surface molecule has the sequence of SEQ ID NO:21. In other aspects, the fourth cell-surface molecule is a CD99 chimera comprising a CD99 extracellular domain and a heterologous transmembrane domain, wherein the fourth cell-surface molecule lacks CD99 intracellular signaling. In other aspects, the heterologous transmembrane domain has at least a 90% sequence identity with SEQ ID NO:6. In other aspects, the transmembrane domain has the sequence of SEQ ID NO:6. In other aspects, the fourth cell-surface molecule has at least a 90% sequence identity with SEQ ID NO:30. In other aspects, the fourth cell surface molecule has the sequence of SEQ ID NO:30.RUC027WSF2024-223

[0031] The invention provides the cells as disclosed herein, wherein the cells have a reduced or eliminated cell surface leukocyte antigen class I. In some aspects, the leukocyte antigen class I is the human leukocyte antigen class I (HLA-I). In other aspects, the leukocyte antigen class I is the swine leukocyte antigen class I (SLA-I). In other aspects, the cell has a reduced or eliminated cell surface leukocyte antigen class II. In other aspects, the leukocyte antigen class II is the human leukocyte antigen class II (HLA-II). In other aspects, the leukocyte antigen class II is the swine leukocyte antigen class II (SLA-II).

[0032] The invention provides the cells as disclosed herein, wherein the cells are ABO blood group compatible with a subject into which the cell is transplanted. In some aspects, the cell is ABO blood group type O. In some aspects, the cell is rhesus factor (Rh) blood group compatible with a subject into which the cell is transplanted. In some aspects, the cell is Rh-.

[0033] The invention provides cells as disclosed herein, wherein the cells are derived from a primary cell, a comprehensively immune protected (CIP) cell, a hypoimmune cell, a hypoimmune pluripotent (HIP) cell, a HIP cell that is ABO blood group O and Rhesus Factor negative (HIPO-), an induced pluripotent stem cell (iPSC), or an embryonic stem cell (ESC). In some aspects, the cells are selected from the group consisting of a chimeric antigen receptor (CAR) cell, an immune cell expressing an endogenous or exogenous T cell receptor (TCR), an immune cell, an endothelial cell, a dopaminergic neuron, a pancreatic islet cell, a cardiomyocyte, a retinal pigment endothelium cell, a thyroid cell, and a parathyroid cell.

[0034] The invention provides cells as disclosed herein, wherein the cells are from a species selected from the group consisting of a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig. In preferred aspects, the cells are from a human. In other preferred aspects, the cells are from a pig. In some aspects, the pig cell comprises a knockout in one or more of a GGTA1, CMAH, B4GALNT2, or growth hormone receptor (GHR) gene.

[0035] The invention provides cells as disclosed herein, wherein the cells comprise one or more of human Thrombomodulin (TBM), CD46, DAF, EPCR, HO-1, or CD59 transgenes.

[0036] The invention provides cells as disclosed herein, wherein the first cell surface molecule is a CD47 chimera, wherein the CD47 chimera comprises a CD47 extracellular domain bound to a cell surface of the cells by a heterologous transmembrane domain.

[0037] The invention provides methods of treating a disease with the cells as disclosed herein, comprising the step of transplanting the cells into a subject, wherein the cells evadeRUC027WSF2024-223 killing by immune cells. In some aspects, the immune cells are leukocytes, myeloid cells, polymorphonuclear (PMN) cells, macrophages, T cells, cytotoxic T cells, or natural killer (NK) cells. In other aspects, the cells are less susceptible to the subject’s immune rejection when compared to a parental cell. In some aspects, the transplantation step utilizes a singlecell suspension of islets cells.

[0038] In some aspects of the invention, the cells and the subject are not of the same species. In other aspects, the subject is a human. In other aspects, the cells are from a species selected from the group consisting of a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig. In a preferred aspect, the cells are pig cells.

[0039] In some aspects of the invention, the method comprises administering a danger- associated molecular pattern (DAMP) inhibitor, a DAMP pathway inhibitor, or a combination thereof. In other aspects, the DAMP inhibitor, DAMP pathway inhibitor, or the combination thereof are delivered to the subject using separate delivery methods.

[0040] In some aspects of the invention, the method comprises administering cyclosporin A (CsA), dexamethasone, an anti-SIRL-1 antibody, a soluble CD99 chimera, BAY 85-8501, alpha-1 antitrypsin (Al AT), TAK-242, apocynin, idelalisib, CpG-52364, colchicine, or the anti-TLR4 antibody NI-0101. In other aspects, the method comprises administering the combination of an anti-SIRL-1 antibody and BAY 85-8501. In other aspects, the method comprises administering the combination of CsA and BAY 85-8501.

[0041] The invention provides a method of treating a disease with a tissue comprising the cells as disclosed herein, comprising the step of transplanting the tissue into a subject, wherein the cells evade killing by the immune cells. In other aspects, the tissue and the subject are not of the same species. In other aspects, the subject is a human. In other aspects, the tissue is from a species selected from the group consisting of a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig. In preferred aspects, the tissue is a pig tissue.

[0042] The invention provides a method of treating a disease with an organ comprising the cells as disclosed herein, comprising the step of transplanting the organ into a subject, wherein the cells evade killing by the immune cells. In some aspects, the organ and the subject are not of the same species. In other aspects, the subject is a human. In other aspects,RUC027WSF2024-223 the organ is from a species selected from the group consisting of a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig. In preferred aspects, the organ is a pig organ.

[0043] The invention provides a pharmaceutical composition, comprising the cells as disclosed herein and a pharmaceutical excipient. In some aspects, the pharmaceutical composition comprises Colchicine, Idelalisib, or oligonucleotide CpG-52364.

[0044] The invention provides a medicament for treating a disease, comprising the cells as disclosed herein and a pharmaceutical excipient. In some aspects, the cells are selected from the group consisting of a chimeric antigen receptor (CAR) cell, an immune cell expressing an endogenous or exogenous T cell receptor (TCR), an immune cell, an endothelial cell, a dopaminergic neuron, a pancreatic islet cell, a cardiomyocyte, a retinal pigment endothelium cell, a thyroid cell, and a parathyroid cell. In other aspects, the disease is selected from the group consisting of Type I Diabetes, Type II Diabetes, monogenic diabetes, a cardiac disease, a neurological disease, a cancer, an ocular disease, a vascular disease, and an endocrinologic disease.

[0045] The invention provides a genetically-modified non-human animal, comprising a reduced or eliminated endogenous leukocyte antigen class I expression, wherein the genetically-modified non-human animal expresses a human CD200R engager (CD200RE), a human SIRPa engager (SIRPaE), a human Fc Sequestration molecule, and a human CD99 ligand engager (CD99LE). In some aspects, the genetically -modified non-human animal further comprises a reduced or eliminated endogenous leukocyte antigen class II expression. In some aspects, the non-human animal is a monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, or a guinea pig. In preferred aspects, the genetically-modified non-human animal is a pig.

[0046] In other aspects, the genetically-modified non-human animal comprises a knockout in one or more of a GGTA1, CMAH, B4GALNT2, or growth hormone receptor (GHR) gene.

[0047] The genetically-modified non-human animal of any one of claims 97-101, wherein the animal comprises human Thrombomodulin (TBM), CD46, DAF, EPCR, HO-1, or CD59 transgenes. In other aspects, the genetically-modified non-human animal has been modified to eliminate the risk of transmitting porcine endogenous retroviruses (PERV). In someRUC027WSF2024-223 aspects, the risk has been eliminated by CRISPR. In other aspects, the genetically -modified non-human animal is PERV-C negative.

[0048] The invention provides a use of the cells as disclosed herein for transplanting into a subject for treating a disease, wherein the cells evade killing by immune cells.

[0049] The invention provides a use of a tissue comprising the cells as disclosed herein for transplanting into a subject for treating a disease, wherein the cells evade killing by immune cells.

[0050] The invention provides a use of an organ comprising the cells as disclosed herein for transplanting into a subject for treating a disease, wherein the cells evade killing by immune cells.

[0051] The invention provides a use of the cells as disclosed herein for manufacturing a medicament for treating a disease, wherein the cells evade killing by immune cells.

[0052] The invention provides a use of a tissue comprising the cells as disclosed herein for manufacturing a medicament for treating a disease, wherein the cells evade killing by immune cells.

[0053] The invention provides a use of an organ comprising the cells as disclosed herein for manufacturing a medicament for treating a disease, wherein the cells evade killing by immune cells.

[0054] The invention provides a method of killing the cells as described herein in vivo, comprising administering a SIRPa antagonist to a subject into which the cell has been transplanted, wherein the cell expresses a cell-surface SIRPa engager. In some aspects, the SIRPa antagonist comprises an immunoglobulin domain. In other aspects, the SIRPa antagonist is an antibody or an scFv. In other aspects, the SIRPa antagonist is an antibody. In other aspects, the antibody binds to the SIRPa with an affinity measured by its dissociation constant (Kd), wherein the Kd is between about 107and 1013M. In other aspects, the antibody has at least a 90% sequence identity to SEQ ID NO:46. In other aspects, the antibody has the sequence of SEQ ID NO:46. In other aspects, the subject is a human. In other aspects, the cell-surface SIRPa engager comprises CD47, a truncated CD47, a CD47 chimera, or an anti-SIRPa immunoglobulin domain.RUC027WSF2024-223V. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1: CD200R is an inhibitory receptor on polymorphonuclear cells (PMNs), macrophages, and some additional immune cells. The CD200R Engager is a synthetic molecule expressed on engineered cells. The CD200R Engager binds to CD200R and exerts agonistic function, which results in an inhibitory signal in that immune cell.

[0056] Figure 2A shows flow cytometry analysis of CD200R Engager expression in transduced wild-type (WT) human iPSC-derived endothelial cells (iECs). Figure 2B shows the CD200R Engager expression in B2M- / - CIITA- / - (DKO) SIRPa-E (SIRPa engager) iECs. These cells are HLA class I and Il-deficient (double knockout; DKO) and express the SIRPa Engager. The lentiviral particles for transduction of the CD200R Engager transgene also included a Red Fluorescent Protein (RFP) tag. The flow cytometry histogram in both panels show RFP expression in untransduced WT iECs or DKO SIRPa-E iECs (left peak) and transduced WT iECs +CD200R-E or transduced DKO SIRPa-E iECs +CD200R-E (right peak). This confirmed their expression of the CD200R Engager.

[0057] Figure 3: BLI killing assay over 2 hours with freshly isolated human polymorphonuclear cells (PMNs). Human Wildtype iECs (WT iECs) express Firefly luciferase for the BLI killing assays. Human WT iECs (Figure 3A) or human WT iECs expressing the CD200R Engager (Figure 3B) were plated and let attach for 16 hours. Then, human PMNs or PMNs together with sonicated cell debris (DAMPs) of the same target cells were added to the wells and let incubate for 2 hours. The percent BLI signal after the 2-hour incubations is shown. PMNs alone did not cause any target cell killing. When DAMPs were added, however, WT iECs were partially killed by PMNs. This PMN killing was mitigated when the target cells expressed the CD200R Engager.

[0058] Figure 4: BLI killing assay over 2 hours with freshly isolated human polymorphonuclear cells (PMNs). Firefly luciferase-expressing human DKO SIRPa-E iECs (Figure 4A) or human DKO SIRPa-E iECs expressing the CD200R Engager (Figure 4B) were plated and let attach for 16 hours. Then, human PMNs or PMNs together with sonicated cell debris (DAMPs) of the same target cells were added to the wells and let incubate for 2 hours. The percent BLI signal after the 2 hours incubation is shown. PMNs alone did not cause any target cell killing. When DAMPs were added, however, DKO SIRPa-E iECs were partially killed by PMNs. This PMN killing was mitigated when the target cells expressed the CD200R Engager.RUC027WSF2024-223

[0059] Figure 5: BLI killing assay over 6 hours with human peripheral blood NK cells or human macrophages (MAC). Firefly luciferase-expressing human DKO iECs (Figure 5A) or human DKO iECs expressing the CD200R Engager (Figure 5B) were plated and let attach for 16 hours. Then, human NK cells or MACs were added to the wells and let incubate for 6 hours. The percent BLI signal after the 6 hours incubation is shown. DKO iECs were killed by NK cells or MACs. DKO iECs expressing the CD200R Engager were largely protected from NK cell killing and MAC killing.

[0060] Figure 6 shows endothelial cells killed by PMNs of a Neu5Gc / Neu5 Ac-mismatched species. Induced wt or HIP ECs from human (Figure 6A), and C57BL / 6 mouse, or rhesus monkey (Figure 6B) were plated on the XCelligence platform and were incubated with PMNs from a species with a Neu5 Ac / Neu5Gc mismatch. The PMNs were not stimulated. In all combinations, the target cells were expeditiously killed by xenogeneic PMNs.

[0061] Figure 7 shows that the above phenomenon is xeno-specific. Killing of allogeneic iECs by unstimulated PMNs is usually not observed. Rhesus monkey ECs were not killed by unstimulated allogeneic rhesus monkey PMNs. Allogeneic PMNs stimulated with DAMPs or macaque IL-2 stimulated allogeneic PMNs, however, did induce killing.

[0062] Figure 8 shows that a CMAH transgene alone did not mitigate human iEC susceptibility to rhesus monkey PMN killing. Human rhHIP iECs (B2M- / - CIITA- / - with rhesus CD47 expression) were transduced to express the missing CMAH using lentiviral particles. These human rhHIP CMAH tg iECs were then challenged with rhesus monkey PMNs on the XCelligence platform. CMAH replacement alone did not alleviate xenogeneic PMN killing.

[0063] Figure 9 shows that CMAH expression combined with CD200 and CD99 overexpression in human rhHIP iECs mitigated rhesus monkey PMN killing. CMAH transduction was combined with overexpression of CD200 and CD99 using lentiviral particles. When they were challenged with rhesus monkey PMNs, target cell killing was prevented. This was observed even when the PMNs were stimulated with sonicated cells (DAMPs) or macaque IL-2.

[0064] Figure 10 shows an exemplary strategy for engineering cells for xenogeneic transplantation, in this case pig cells. They have knocked out swine leukocyte antigen (SLA)- I and SLA-II function or expression, a SIRPa Engager (or CD47 chimera, here exemplified as truncated CD47) that does not carry any CD47 intracellular signalling motifs (eitherRUC027WSF2024-223 through the transmembrane domain or the intracellular domain), as well as a truncated CD64 extracellular domain. In addition, the engineered cells express human CD99 and CD200.

[0065] Figure 11A shows pig wt endothelial cells (ECs) that were incubated with human PMNs. The PMNs were either unstimulated or stimulated with cell debris (DAMPs) or human IL-2. The pig target cells were transduced to express firefly luciferase and their bioluminescence signal (BLI signal) was recorded over time. The relative BLI signal after 24 hours is plotted in the graph. Human PMNs, whether unstimulated or stimulated, killed the pig wt target cells.

[0066] Figure 11B shows pig wt ECs that were incubated with primed human PBMCs. The human PBMCs were primed by incubating them with pig ECs for 10 days in vitro. Then, PBMCs were sorted by CD45 expression to exclude the pig ECs and enrich primed human PBMCs. The primed PBMCs were then added to the pig wt ECs. After 24 h, the pig wt EC target cells were killed.

[0067] Figure 11C shows 50,000 firefly luciferase-positive pig wt ECs injected into xenogeneic humanized mice together with 1 million human PMNs and 1 mg anti -MICA IgGl antibody. On days 1 and 2, another dose of 1 mg anti -MIC A IgGl antibody was injected into the EC grafts. Cell survival was assessed by serial bioluminescence imaging. As shown, all 4 pig wt EC grafts were quickly killed and their BLI signals vanished in just 2 days.

[0068] Figure 12A shows pig wt ECs engineered to express human CD99 and CD200 (wt.99.200 ECs) that were not killed by unstimulated or stimulated human PMNs. Figure 12B shows that pig wt ECs engineered to express human CD99 and CD200 (wt.99.200 ECs) were killed by primed human PBMCs. Assays were run over 24 hours.

[0069] Figure 13A shows that pig ECs with depleted SLA class I and II but expressing a truncated CD47.140a chimera, CD99, and CD200 (B2m- / - Ciita- / -.tCD47.99.200) were not killed by PMNs. Figure 13B shows that when these ECs were incubated with primed human PBMCs, the engineered pig target cells survived and the BLI signal stayed stable. Assays were run over 24 hours.

[0070] Figure 13C shows B2m- / - Ciita- / -.tCD47.99.200 ECs incubated with a cytotoxic anti- MICA IgGl antibody and human NK cells were killed. Pig B2m' / 'Ciita' / " .tCD47.tCD64.99.200 ECs were engineered to also express the truncated CD64.140a Fc receptor.RUC027WSF2024-223

[0071] Figure 14A shows that these pig ECs with depleted SLA class I and II but expressing a truncated CD47.140a chimera, CD99, CD200, and a truncated CD64.140a Fc receptor are not killed by unstimulated and stimulated human PMNs. Figure 14B shows that these ECs were not killed by primed human PBMCs. Figure 14C shows that these ECs were not killed by cytotoxic anti-MICA IgGl antibody and human NK cells. Assays were run over 24 hours.

[0072] Figure 14D shows 50,000 firefly luciferase-positive pig B2m' / 'Ciita' / ‘ .tCD47.tCD64.99.200 ECs injected into xenogeneic humanized mice together with 1 million human PMNs and 1 mg anti-MICA IgGl antibody. On days 1 and 2, another dose of 1 mg anti-MICA IgGl antibody was injected into the EC grafts. Cell survival was assessed by serial bioluminescence imaging. As shown, all four pig B2m' / 'Ciita' / '.tCD47.tCD64.99.200 EC grafts survived for at least about 40 days. Their BLI signals remained stable and even increased over time suggesting continued proliferation of the ECs.

[0073] Figure 15 shows wild-type, B2M- / - CIITA- / - SIRPaE.140a CD64.140a and B2M- / - CIITA- / - tCD47.140a CD64.140a islet cell phenotypes characterized by flow cytometry. HLA-I, HLA-II, SIRPaE, CD47, and CD64 were measured on the cell surface.

[0074] Figure 16 shows that the engineered islet cells of Figure 15 did not show impeded endocrine competence. They sensed glucose and release insulin.

[0075] Figure 17 shows wild-type islet cells, B2M- / - CIITA- / - SIRPaE.140+ CD64.140a+, or B2M- / - CIITA- / - tCD47.140+ CD64.140a+ islet cells injected into 3 humanized mice each. After 6 days, the spleens were recovered and splenocytes were isolated for ELISpot assays and serum was recovered for the detection of antibodies. The wild-type group evoked a strong T cell response. In contrast, both engineered comprehensively immune protected (CIP) islets did not show any T cell activation above background.

[0076] Figure 18 shows flow cytometry assessment of donor cell-specific antibodies generated in the above mice 6 days after they were injected with one of the WT or CIP islet cells. Sera from the mice were incubated with the same islet cell fraction that was injected. A secondary anti-human antibody was used to detect antibody bound to the islet cells. We found donor islet-specific antibody binding to the wild-type islet cells. There was no antibody-binding in both CIP islet groups.

[0077] Figure 19 shows wild-type (WT), DKO (B2M- / - CIITA- / -) islet cells that were HLA class I and II deficient but did not express any transgenes, or CIP islet cells (B2M- / - CIITA- / - SIRPaE.140+ CD64.140a+ or B2M- / - CIITA- / - tCD47.140+ CD64.140a+) that wereRUC027WSF2024-223 incubated with allogeneic primary human NK cells or macrophages. The wild-type cells remained unaffected by NK cells and macrophages. The DKO islets were killed expeditiously by NK cells and macrophages. Both CIP islets, however, resisted innate immune cell killing.

[0078] Figure 20 shows that WT islet cells transplanted into allogeneic, diabetic humanized mice get rejected as their BLI signal vanishes over approximately 10 days. These WT islets did not affect fasting glucose levels and did not treat diabetes. In contrast, both engineered (CIP) islet populations survived in allogeneic, diabetic humanized mice and affected reduced glucose levels. This indicated diabetes mitigation.

[0079] Figure 21 shows that only the CIP islets (expressing CD64.140a) resisted ADCC and CDCC in vitro. Islet cells were challenged with anti-EGFR antibodies (cetuximab) and either NK cells or serum. All islet cells not expressing CD64.140a were quickly killed.

[0080] Figure 22 shows that CIP islet cells resisted ADCC and CDCC in vivo. Five different islet cell populations were transplanted into allogeneic, diabetic humanized mice. On days 0, 1, and 2, all groups received 3 doses of Img cetuximab intramuscularly into the grafts. On day 0, the animals additionally received one million human NK cells. Only the islets expressing CD64.140a were fully protected from antibody-mediated rejection. They survived and were able to treat diabetes.

[0081] Figure 23A shows CDC killing assays with serum from two patients with type 1 diabetes mellitus that did not have detectable Znt8 antibodies. CIP cells avoid CDC killing. All three islet cells, WT islet cells, B2M- / - CIITA- / - tCD47.140a+ islet cells, and and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ islet cells (CIP), survived in this assay. Serum from a patient with type 1 diabetes mellitus having 11 pg / ml of Znt8 antibody levels of (just above the 8 pg / ml test threshold) killed WT islet cells and B2M- / - CIITA- / - tCD47.140a+ islet cells (Figure 23B). Only B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ (CIP) islet cells survived the CDC assay. Serum from two patients with type 1 diabetes mellitus having 98 pg / ml and 201 pg / ml Znt8 antibody levels (high antibody titers) showed rapid CDC killing of WT islet cells and B2M- / - CIITA- / - tCD47.140a+ islet cells (Figure 23C). Surprisingly, B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ (CIP) islet cells were fully protected from even these high titers.

[0082] Figure 24 shows wild-type (WT), B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+ (CIP) and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ (CIP) stem cell-derived islet cell phenotypes characterized by flow cytometry. HLA-I, HLA-II, CD47, SIRPaE, and CD64RUC027WSF2024-223 were measured on the cell surface. WT islet cells showed HLA class I expression as the population stained with HLA class I antibody clearly separated from the population stained with isotype control antibody. WT cells were further largely negative for HLA class II and expressed endogenous CD47 levels. They did not express the SIRPaE.140a or CD64.140a transgenes. The B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ (CIP) stem cell-derived islet cells were negative for HLA class I and II and expressed the tCD47.140a and CD64.140a transgenes. The B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+ (CIP) stem cell-derived islet cells were negative for HLA class I and II, expressed endogenous CD47 levels, and the SIRPaE.140a and CD64.140a transgenes.

[0083] Figure 25 shows the insulin release of clustered stem cell-derived islets in vitro in 5.8 mM glucose medium. The engineering to generate CIP stem cell-derived islets (B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ or B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+) did not impede the endocrine competence of the islet cells to sense glucose and release insulin.

[0084] Figure 26: Transplantation of stem cell-derived islets into humanized mice. Six days before injection of 1000 stem cell-derived islets, humanized mice received intraperitoneal Streptozotocin (STZ) injections (60 mg / kg; Sigma-Aldrich Cat. No. 572201). Blood was regularly drawn after transplantation and blood glucose was measured. In addition, all islet grafts were firefly luciferase positive and graft survival was followed using BLI imaging. Wild-type (WT) stem cell-derived islet cell grafts were rejected within 10 days in allogeneic, diabetic humanized mice (Figure 26A). Blood glucose levels, which were >400 mg / dl at the time of transplantation, did not improve and remained steadily high. In contrast, both hypoimmune CIP stem cell-derived islet grafts (B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ or B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+) survived in their allogeneic hosts as indicated by the stable BLI signals during follow up. Also, both hypoimmune CIP islet cell populations were able to lower blood glucose levels over time, almost reaching levels of healthy animals. (Figures 26B and 26C).

[0085] Figure 27A shows EGFR expression on all stem cell-derived islet cells. EGFR was constitutively expressed in WT stem cell-derived islet cells and engineered stem cell-derived islet cells. Figure 27B shows anti-EGFR IgGl antibody (cetuximab) used to induce in vitro killing by either NK cells (antibody-mediated cellular cytotoxicity, ADCC) or complement (complement-dependent cytotoxicity, CDC). All stem cell-derived islet cells showed stable growth in vitro without intervention. When cetuximab with NK cells or cetuximab with serum was added, the WT islets were killed quickly. In contrast, the fully engineered stemRUC027WSF2024-223 cell-derived CIP islets (B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+ and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+) were fully protected against antibody-mediated killing.

[0086] Figure 28 shows CIP islet cells avoid antibody-mediated killing in vivo. WT islets, B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+, and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ CIP islets were injected into diabetic NSG mice. With the injection, they received 1 mg of cetuximab and one million human NK cells. On days 1 and 2, another 1 mg cetuximab was injected into the initial islet graft sites to ensure a very high antibodyconcentration in the immediate vicinity of the grafts. All grafts were positive for firefly luciferase and graft survival was monitored by BLI imaging. Blood glucose was followed to assess the ability of the islet grafts to treat diabetes. The WT stem cell-derived islet grafts were rejected over a few days and had no impact on blood glucose levels (Figure 28A). Both fully engineered CIP islets (B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+ and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+), however, were protected against antibody-mediated killing, showed stable BLI signals during follow-up, and had normalized blood glucose levels at the end of the study period (Figures 28B and 28C).

[0087] Figure 29A shows unedited, WT pig pancreatic islet cells (ICs) incubated with primed human xenogeneic T cells. Prior to that, the T cells had been cultured with WT pig ICs for 10 days and then T cells were sorted and added to this assay. WT pig ICs were further incubated with human IL-2-stimulated NK cells and macrophages. Pig WT ICs were killed by primed T cells but not by NK cells or macrophages. Figure 29B shows WT pig ICs incubated with 1 pg / ml cetuximab and either human serum or human NK cells. The pig WT ICs were killed via both CDC and ADCC assay. Figure 29C shows WT pig ICs incubated with human PMNs. The PMNs were either unstimulated or stimulated with cell debris (DAMPs) or human IL-2. Human PMNs, whether unstimulated or stimulated, killed the pig WT ICs. The pig target cells were transduced to express firefly luciferase and their bioluminescence signal (BLI signal) was recorded over time. The relative BLI signal after 24 hours is shown.

[0088] Figure 30A shows pig DKO ICs incubated with primed human T cells. Pig DKO ICs were further incubated with human IL-2-stimulated NK cells and macrophages. Pig DKO ICs were killed by NK cells and macrophages but not by primed T cells. Figure 30B shows pig DKO ICs incubated with 1 g / ml cetuximab and either human serum or human NK cells. The pig DKO ICs were killed via both CDC and ADCC pathways. Figure 30C shows pig DKO ICs incubated with human PMNs. The PMNs were either unstimulated or stimulated with cellRUC027WSF2024-223 debris (DAMPs) or human IL-2. Human PMNs, whether unstimulated or stimulated, killed the pig DKO ICs. The pig target cells were transduced to express firefly luciferase, and their bioluminescence signal (BLI signal) was recorded over time. The relative BLI signal after 24 hours is shown.

[0089] Figure 31A shows pig DKO tCD47.140a+ CD64.140a+ ICs (CIP) incubated with primed human T cells, human IL-2-stimulated NK cells, and macrophages. Pig DKO tCD47.140a+ CD64.140a+ ICs were protected from primed T cells, NK cells, and macrophages. Figure 31B shows pig DKO tCD47.140a+ CD64.140a+ ICs incubated with 1 pg / ml cetuximab and either human serum or human NK cells. They were protected from both CDC and ADCC killing. Figure 31C shows pig DKO tCD47.140a+ CD64.140a+ ICs incubated with human PMNs. The PMNs were either unstimulated or stimulated with cell debris (DAMPs) or human IL-2. Human PMNs, whether unstimulated or stimulated, killed these pig ICs. The pig target cells were transduced to express firefly luciferase, and their bioluminescence signal (BLI signal) was recorded over time. The relative BLI signal after 24 hours is shown.

[0090] Figure 32A shows pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 ICs incubated with primed human T cells, human IL-2-stimulated NK cells, and macrophages. Pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 ICs were protected from primed T cells, NK cells, and macrophages. Figure 32B shows pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 ICs incubated with 1 pg / ml cetuximab and either human serum or human NK cells. The pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 ICs were protected from both CDC and ADCC killing. Figure 32C shows pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 ICs incubated with human PMNs. The PMNs were either unstimulated or stimulated with cell debris (DAMPs) or human IL-2. Pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 ICs were protected from human PMNs both unstimulated and stimulated. The pig target cells were transduced to express firefly luciferase, and their bioluminescence signal (BLI signal) was recorded over time. The relative BLI signal after 24 hours is shown.

[0091] CD99 and CD200 were then exchanged for the chimeras CD99.140a and CD200.140a. Figure 32D shows pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs incubated with primed human T cells, human IL-2-stimulated NK cells, and macrophages. Pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs were protected from primed T cells, NK cells, and macrophages. Figure 32E shows pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs incubated with 1 pg / mlRUC027WSF2024-223 cetuximab and either human serum or human NK cells. The pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs were protected from both CDC and ADCC killing. Figure 32F shows pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs incubated with human PMNs. The PMNs were either unstimulated or stimulated with cell debris (DAMPs) or human IL-2. Pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs were protected from human PMNs both unstimulated and stimulated. The pig target cells were transduced to express firefly luciferase and their bioluminescence signal (BLI signal) was recorded over time. The relative BLI signal after 24 hours is shown.

[0092] Figure 33A shows pig WT islet transplantation into diabetic, xenogeneic humanized mice. Five hundred thousand islet cells were mixed with one million human PMNs and were injected into the hindlimb muscle. Islets were transduced to express firefly luciferase and their survival was monitored through BLI (upper graph). All pig WT islet grafts were rejected within 10 days as shown by the drop of the BLI signal into the background (dashed line). Islets were injected 6 days after the mice received Streptozotocin (STZ) and all animals showed fasting blood glucose levels > 400 mg / dL (lower graph). The islet transplantation did not affect fasting blood glucose levels, and all animals remained diabetic. Figure 33B shows pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 islet transplantation into diabetic, xenogeneic humanized mice. Five hundred thousand islet cells were mixed with one million human PMNs and were injected into the hindlimb muscle. Islets were transduced to express firefly luciferase and their survival was monitored through BLI (upper graph). All pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 islet grafts survived for at least about 50 days post transplantation and the increased BLI signal indicates an increase in metabolic activity over time. This shows improved vascularization. Islets were injected 6 days after the mice received STZ and all animals showed fasting blood glucose levels > 400 mg / dL (lower graph). In all mice, pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 islet transplantation normalized fasting blood glucose levels by about 10 days post-transplantation and remained normalized at about 200 mg / dl and remained stable for at least about 50 days.

[0093] Figure 33C: CD99 and CD200 were exchanged for the chimeras CD99.140a and CD200.140a. The protocols were the same as in Figure 33B except pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs was used. Five hundred thousand firefly luciferase-positive islet cells were mixed with one million human PMNs and were injected into the hindlimb muscle. All pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ islet grafts survived for at least about 50 days post transplantation (upperRUC027WSF2024-223 graph). Islets were injected 6 days after the mice received STZ and all animals showed fasting blood glucose levels > 400 mg / dL (lower graph). In all mice, pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ islet transplantation normalized fasting blood glucose levels by about 10 days post-transplantation and remained normalized at about 200 mg / dl and remained stable for at least about 50 days.

[0094] Figure 34A shows human WT parathyroid (PTH) cells incubated with primed allogeneic human T cells, human IL-2-stimulated NK cells, or macrophages. The PTH cells were transduced to express firefly luciferase. After 24 hours, the change in BLI signal was recorded. WT PTH cells were killed by primed, allogeneic T cells, but were protected from NK cell and macrophage killing. Figure 34B shows human WT PTH cells incubated with cetuximab and human serum or human NK cells. The change in BLI signal is shown after 24 hours of incubation. WT PTH cells were killed by serum through CDC and by NK cells through ADCC killing.

[0095] Figure 35A shows human DKO PTH cells (B2M- / - CIITA- / -) incubated with primed allogeneic human T cells, human IL-2-stimulated NK cells, or macrophages. The PTH cells were transduced to express firefly luciferase. After 24 hours, the change in BLI signal was recorded. DKO PTH cells were protected from primed, allogeneic T cells, but were killed by NK cells and macrophages. Figure 35B shows human DKO PTH cells incubated with cetuximab and human serum or human NK cells. After 24 hours, the change in BLI signal was recorded. DKO PTH cells were killed by serum through CDC and by NK cells through ADCC killing.

[0096] Figure 36A shows human CIP PTH cells (DKO tCD47.140a+ CD64.140a+) incubated with primed allogeneic human T cells, human IL-2-stimulated NK cells, or macrophages. The PTH cells were transduced to express firefly luciferase. After 24 hours, the change in BLI signal was recorded. Hypo PTH cells were protected from primed, allogeneic T cells, NK cells, and macrophages. Figure 36B shows human CIP PTH cells incubated with cetuximab and human serum or human NK cells. After 24 hours, the change in BLI signal was recorded. CIP PTH cells were protected from both CDC and ADCC killing.Figure 37A shows the fate of firefly luciferase-expressing human WT PTH cells after transplantation into humanized mice. The WT grafts were rejected within 7 to 10 days when the BLI signal dropped into the background. Figure 37B shows the survival of firefly luciferase-expressing human CIP PTH cells (DKO tCD47.140a+ CD64.140a+) after transplantation into humanized mice. All grafts maintained stable BLI signal over the studyRUC027WSF2024-223 period of 29 days, which indicates unobstructed graft survival. Three mice per group, all individual mice are shown.

[0097] Human primary islet cells DKO tCD47.140a, DKO tCD47.140a.ini (SEQ ID NO: 40), or DKO tCD47.140a.ms (SEQ ID NO: 41) were generated and re-clustered. A total of 450 firefly luciferase-positive islet clusters were injected intramuscularly into diabetic, allogeneic humanized mice. Figure 38A shows that DKO tCD47.140a islet grafts survived for at least about 30 days in allogeneic humanized mice and alleviated diabetes. Figure 38B shows similar survival and efficacy for DKO tCD47.140a.ini islet grafts. Figure 38C shows similar survival and efficacy for DKO tCD47.140a.ms islet grafts.

[0098] Figure 39 shows that unedited (WT), primary human islets were not susceptible to IL-2-stimulated NK cell killing or ADCC using escalating doses of a human aSIRPa.

[0099] Figure 40 shows that DKO SIRPaE.140a CD64.140a primary human islets were also not susceptible to IL-2-stimulated NK cell killing but were killed when a human aSIRPa was added to the NK cells. Higher doses led to faster killing.

[0100] Figure 41 shows that DKO tCD47.140a CD64.140a primary human islets were also not susceptible to IL-2-stimulated NK cell killing but were killed when a human aSIRPa was added to the NK cells. Higher doses led to faster killing.

[0101] Figure 42 shows primary human islet killing in humanized mice. Six days before the transplantation (day -6), mice received streptozotocin (STZ) and became diabetic by day 0. Firefly luciferase-positive DKO SIRPaE.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice. On day 0, human NK cells and aSIRPa was administered (see arrows) and two more doses of aSIRPa were given on days 1 and 2. The islet graft vanished (upper panel) and all mice remained diabetic (lower panel).

[0102] Figure 43 shows primary human islet killing in humanized mice. Six days before the transplantation (day -6), mice received STZ and became diabetic by day 0. Firefly luciferase-positive DKO tCD47.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice. On day 0, human NK cells and aSIRPa was administered (see arrows) and two more doses of aSIRPa were given on days 1 and 2. The islet graft vanished (upper panel), and all mice remained diabetic (lower panel).

[0103] Figure 44 shows human islet killing in humanized mice. Six days before the transplantation (day -6), mice received STZ and became diabetic by day 0. Firefly luciferase-RUC027WSF2024-223 positive DKO tCD47.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice. On day 7, human NK cells and aSIRPa was administered (see arrows) and two more doses of aSIRPa were given on days 8 and 9. The islet cells engrafted initially and then vanished after day 7 (upper panel), and although fasting glucose levels were initially decreasing, all mice became diabetic again after day 7 (lower panel).

[0104] Figure 45 shows that unedited (WT), primary pig islets were not susceptible to human IL-2 stimulated NK cell killing or ADCC using escalating doses of a human aSIRPa.

[0105] Figure 46 shows that DKO tCD47.140a CD64.140a primary pig islets were also not susceptible to IL-2 stimulated NK cell killing but were killed when a human aSIRPa was added to the NK cells. Higher doses led to faster killing.

[0106] Figure 47 shows that DKO tCD47.140a CD64.140a CD99 CD200 primary pig islets were not susceptible to IL-2 stimulated NK cell killing but were killed when a human aSIRPa was added to the NK cells. Higher doses led to faster killing.

[0107] Figure 48 shows pig islet killing in humanized mice. Six days before the transplantation (day -6), mice received STZ and became diabetic by day 0. Firefly luciferasepositive DKO tCD47.140a CD64.140a CD99 CD200 primary pig islets were transplanted into diabetic, allogeneic humanized mice additionally receiving PMNs with the graft on day 0. On day 30, human NK cells and aSIRPa was administered (see arrows) and two more doses of aSIRPa were given on days 31 and 32. The islet grafts, which had been stable until day 30, vanished (upper panel), and all mice, which had reached fasting glucose levels of around 200 mg / dl, became diabetic again (lower panel).

[0108] Figure 49 shows primary human islet killing in humanized mice. Six days before the transplantation (day -6), mice received STZ and became diabetic by day 0. Firefly luciferase-positive DKO tCD47.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice. On day 0, human SIRPa- / - NK cells were administered (see arrows). The islet graft vanished (upper panel), and all mice remained diabetic (lower panel).

[0109] Figure 50 shows primary human islet killing in humanized mice. Six days before the transplantation (day -6), mice received STZ and became diabetic by day 0. Firefly luciferase-positive DKO tCD47.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice. On day 7, human SIRPa- / - NK cells were administeredRUC027WSF2024-223(see arrows). These islet grafts, which had already engrafted, vanished (upper panel) and all mice became diabetic again (lower panel).

[0110] Figure 51 shows primary pig islet killing in humanized mice. Six days before the transplantation (day -6), mice received STZ and became diabetic by day 0. Firefly luciferase-positive DKO tCD47.140a CD64.140a CD99 CD200 primary pig islets were transplanted into diabetic, allogeneic humanized mice additionally receiving PMNs with the graft on day 0. On day 30, human SIRPa- / - NK cells were administered (see arrows). The islet grafts, which had been stable until day 30, vanished (upper panel), and all mice, which had reached fasting glucose levels of around 200 mg / dl, became diabetic again (lower panel).

[0111] Figure 52A shows the BLI imaging graphs for the survival of human DKO tCD47.140a CD64.140a islet cells in single cell suspension (top) or after re-clustering (bottom) into diabetic NSG mice. No differences in survival were observed.

[0112] Figure 52B shows the function of the transplanted islet cells in Figure 52A to alleviate diabetes in diabetic NSG mice. Six days before the injection of islet cells, mice received intraperitoneal Streptozotocin (STZ) injections (60 mg / kg; Sigma-Aldrich Cat. No. 572201). Blood was regularly drawn before and after transplantation and blood glucose was measured. The islet cells that were transplanted in single cell suspension are shown on top and those transplanted after re-clustering are shown on the bottom. Both groups were similarly effective in alleviating diabetes and no differences between groups were seen.

[0113] Figure 53A shows the BLI imaging graphs for the survival of human DKO tCD47.140a CD64.140a islet cells at a concentration of 50M per ml. Cells were transplanted in single cell suspension (top) or after re-clustering (bottom) into diabetic NSG mice. No differences in survival were observed.

[0114] Figure 53B shows the function of the above transplanted islet cells to alleviate diabetes in diabetic NSG mice. Six days before the injection of islet cells, mice received intraperitoneal Streptozotocin (STZ) injections (60 mg / kg). Blood was regularly drawn before and after transplantation and blood glucose was measured. The islet cells that were transplanted in single cell suspension are shown on top and those transplanted after reclustering are shown on the bottom. Both groups were similarly effective in alleviating diabetes and no differences between groups were seen.

[0115] Figure 54A shows a representative picture of an islet cluster that formed spontaneously in vivo after injection of a single islet cell suspension at 25M per ml. TheRUC027WSF2024-223 clusters were approximately 100 pm in diameter. Figure 54B shows a representative picture of an islet cluster that formed spontaneously in vivo after injection of single cells in suspension at 50M per ml. The spontaneous islet clusters were again approximately 100 pm in diameter.VI. DETAILED DESCRIPTION OF THE INVENTIONA. Introduction

[0116] The invention provides therapeutic cells that are comprehensively immune protected having a reduced or eliminated surface leukocyte antigen class I, an enhanced SIRPa engager (SIRPaE) expression, and an enhanced Fc sequestration molecule expression, (e.g. a CD64 ECD). The cell-surface proteins are combined with a transmembrane domain having structural simplicity, no intracellular signalling, stable expression, and broad compatibility. They also do not have intracellular domains (ICD) or have heterologous ICDs that do not carry out their cognate intracellular signalling. The invention further provides CIP cells, tissues, and organs that are suitable for xenogeneic transplantation. These cells further comprise a CD200R engager and a CD99L engager.

[0117] Thus, the invention provides modified cells that have an increased immune cell resistance. The cells mitigate adaptive and innate immunity when transplanted into a subject when compared to a parental cell. In some aspects of the invention, the cells resist host cell immunity because they are Comprehensively Immune Protected (CIP) cells. CIP cells as disclosed herein have a reduced or eliminated MHC-I complex (in humans HLA-I complex), a reduced or eliminated MHC-II complex (in humans HLA-II complex), a SIRP-a engager molecule on the cell surface, and an antibody Fc sequestration molecule on the cell surface. Examples of a SIRP-a engagers include engineered molecues that have a CD47 extracellular domain or an immunoglobulin domain that binds to a SIRP-a on host macrophages or NK cells. Examples of Fc sequestration molecules include engineered molecules that have a CD64, CD16, or CD32 extracellular domain. To prevent intracellular signalling by the engineered molecules, they do not retain their native intracellular domains.

[0118] In some embodiments, the engineered molecules comprise a heterologous transmembrane domain. Transmembrane domains are known in the art but engineering the molecules with the correct transmembrane domain was an important advance.RUC027WSF2024-223

[0119] One such transmembrane domain is that from CD140a. The CD140a transmembrane domain benefited the invention in several ways. 1) It is a single-pass alphahelical segment. When combined with the ECDs of the invention without an intracellular domain (ICD), it avoided ligand-independent or constitutive signaling. The TMD itself lacks kinase activity or other signaling motifs. Without being bound by theory, the CD140a transmembrane domain acted as a simple linker or tether for anchoring extracellular domains to the membranes without unwanted signalling funci tons. Lacking an ICD prevented downstream signaling cascades (e.g., autophosphorylation or effector recruitment). This feature thus increased surface retention without intracellular signaling. 2) The CD 140a TMD allowed high levels of cell surface expression and prolonged half-life. 3) The CD140a TMD allowed dimerization or oligomerization of the ECDs in the membrane. This enhanced the stability and avidity of the ECD chimeras. 4) Its hydrophobicity increased compatibility with the lipid bilayers and chimeric architectures in different cell types.

[0120] Thus, the invention contemplates cell-surface SIRP-a engager domains (e.g. CD47 ECD) and cell-surface Fc sequestration domains (e.g. a CD64 ECD) combined with a transmembrane domain having structural simplicity, no intracellular signalling, stable expression, and broad compatibility. The invention further contemplates TMDs from, e.g., CD8a, CD4, CD140b, CD8b, CD19, CD28, CD137, CD80, CD178, CD3g, CD3d, CD45, CD44, CD31, CD235a, CD29, CD54, CD106, CD102, and CD58.

[0121] The invention also contemplates cells suitable for xenogeneic transplantation. Such cells additionally express on their cell surface a ligand for CD200R (e.g. CD200) and a receptor for a CD99 ligand (CD99L) (e.g. CD99). In some embodiments, these cell-surface molecules lack intracellular signalling - either through an intracellular domain or a transmembrane domain.

[0122] Xenotransplantation is a promising strategy to alleviate the shortage of organs for human transplantation. In addition to the concerns about pig-to-human immunological compatibility, the risk of cross-species transmission of porcine endogenous retroviruses (PERVs) has impeded the clinical application of this approach. The invention contemplates the elimination of PERV activity in pigs and cells derived therefrom. In some embodiments, CRISPR-Cas9 is used to inactivate PERVs in pigs and cells derived therefrom. (Niu D et al., Science, 357(6357): 1303-1307 (2017). doi: 10.1126 / science.aan4187. Epub 2017 Aug 10. PMID: 28798043; PMCID: PMC5813284, incorporated by reference herein in its entirety.)RUC027WSF2024-223

[0123] In other embodiments, the cells of the invention are hypoimmune cells or hypoimmune pluripotent (HIP) cells. In other embodiments, the cells are Comprehensively Immune Protected (CIP) cells. In other embodiments, the cells are blood type O (HIPO), Rhesus factor (Rh) negative (HIP-) or both type O and Rh- (HIPO-). In other embodiments, the cells have been derived or differentiated from induced stem cells, pluripotent stem cells, embryonic stem cells, HIP, HIP-, or HIPO- cells. In other embodiments, the cells are derived from non-pluripotent cells.

[0124] The invention provides therapeutic cells that engage CD200R and CD99L on immune cells. This reduces sensitivity to a recipient's immune system that kills the therapeutic cells during the window of time shortly after transplantation. In some embodiments, the therapeutic cells are used in a regenerative cell therapy for treating injured organs and tissue. In some embodiments, the regenerative cell therapy of the invention utilizes chimeric antigen receptor (CAR) cells, endothelial cells, fibrocytes, dopaminergic neurons, pancreatic islet cells, hepatocytes, cardiomyocytes, retinal pigment endothelium cells, thyroid cells, or parathyroid cells are used for treating diseases or rehabilitating damaged tissues. In some embodiments, the invention blocks PMN cell killing induced by cytokines, chemokines, danger-associated molecular patterns (DAMPs), or pathogen- associated molecular patterns (PAMPs).

[0125] A summary of the PMN inhibitory strategies is shown in Figure 1. In some embodiments, PMNs are inhibited by targeting the CD200 Receptor (CD200R). In other embodiments, transplanted cells such as hypoimmune cells (HIP), HIP pluripotent cells or derivatives thereof, express an anti-CD200R domain on the surface of an engineered cell.

[0126] The inhibitory CD200R engagement strategies provided herein may be operative during the transplantation / xenotransplantation and engraftment period when strong activating PMN signals are present and no further treatment is necessary for maintianiang hypoimmune cells or their derivatives. Alternatively, the inhibitory CD200R engagement strategies may be operative where permanent PMN inhibition is desired.

[0127] In some embodiments of the invention, Hypolmmunogenic Pluripotent (“HIP”) cells, or derivatives thereof, are transplanted in combination with PMN cell- inhibitory strategy. HIP cells avoid host immune responses due to several genetic manipulations. The cells lack major immune antigens that trigger immune responses and are engineered to avoid phagocytosis and NK cell killing. In some embodiments, the HIP cells are made by eliminating the activity of both alleles of a B2M gene in an induced pluripotentRUC027WSF2024-223 stem cell (iPSC); eliminating the activity of both alleles of a CIITA gene in the iPSC; and increasing the expression of CD47 in the iPSC. HIP cells are described in detail in WO2018132783, incorporated by reference herein in its entirety.

[0128] In some embodiments of the invention, Hypolmmunogenic Pluripotent Blood group O Rh - (“HIPO-”) cells, or derivatives thereof, are transplanted in combination with a PMN cell-inhibitory strategy. HIPO- cells avoid host immune responses due to several genetic or enzymatic manipulations. The cells lack major blood group and immune antigens that trigger immune responses and are engineered to avoid rejection, phagocytosis, or killing. This allows the derivation of “off-the-shelf’ cell products for generating specific tissues and organs. The benefit of being able to use human allogeneic HIPO- cells and their derivatives in human patients provides significant benefits, including the ability to avoid long-term adjunct immunosuppressive therapy and drug use generally seen in allogeneic transplantations. They also provide significant cost savings as cell therapies can be used without requiring individual treatments for each patient.

[0129] HIPO- / CD200R engager cells may serve as a universal cell source for the generation of universally-acceptable derivatives. HIPO- cells are described in detail in U.S. Pat. No. 11,162,079 and IntT Pub. No. WO2020 / 231882. In some embodiments, the transplanted cells comprise enhanced CD 16, CD32, or CD64 expression to evade antibodydependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Such cells are described in IntT Pub. No. WO2021 / 076427. The foregoing are incorporated by reference herein in their entirety.

[0130] In some embodiments, the CD200R engager cells further comprise immune checkpoint engager molecules. Such Immune checkpoint engager molecules may engage, for example, SIRPa, PD-1, TIM3, LILRB3, or LILRB1. Such immune checkpoint engager molecules are described, for instance, in WO2022 / 125439 and W02023 / 200796. The foregoing are incorporated by reference herein in their entirety.

[0131] Thus, the invention provides a cell, comprising a reduced or eliminated MHC- I (HLA-I), a cell surface SIRP-a engager lacking intracellular signalling, and a cell surface antibody Fc sequestration molecule lacking intracellular signalling. The xenogeneic versions of the cells further comprise a cell surface CD200R engager molecule lacking intracellular signalling and a cell surface CD99L engager molecule lacking intracellular signalling. Such cells showed a surprising resilience and complete protection from host cell immune rejection.RUC027WSF2024-223B. Definitions

[0132] The term “about” is given its ordinary and customary meaning. In some aspects, the term means “approximately” or “close to but not exactly.” This meaning may be applied, for example, to numerical values. In some aspects, the term “about” may be applied to a numerical value + / - 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% of the numerical value. In preferred aspects, “about” is the numerical value + / -10%.

[0133] The terms “chimera” or “chimeric protein” refer to a protein that has two or more different protein fragments in a fusion protein. These fragments may come from different genes, different species, or even different parts of the same protein. Likewise, a “chimeric gene” refers to a gene that is formed by combining two or more different genetic coding sequences. These sequences may come from different proteins, different species, or even different parts of the same gene.

[0134] The term “pluripotent cells” refers to cells that can self-renew and proliferate while remaining in an undifferentiated state and that can, under the proper conditions, be induced to differentiate into specialized cell types. The term “pluripotent cells,” as used herein, encompass embryonic stem cells and other types of stem cells, including fetal, amnionic, or somatic stem cells. Exemplary human stem cell lines include the H9 human embryonic stem cell line. Additional exemplary stem cell lines include those made available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (as described in Cowan, C. A. et. al, New England J. Med. 350: 13. (2004), incorporated by reference herein in its entirety.)

[0135] “Pluripotent stem cells” as used herein have the potential to differentiate into any of the three germ layers: endoderm (e.g. the stomach linking, gastrointestinal tract, lungs, etc), mesoderm (e.g. muscle, bone, blood, urogenital tissue, etc) or ectoderm (e.g. epidermal tissues and nervous system tissues). The term "pluripotent stem cells," as used herein, also encompasses “induced pluripotent stem cells”, or “iPSCs”, a type of pluripotent stem cell derived from a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the induction of iPS cells are known in the art and are further described 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 el al., Cell Stem Cell 8:381-384 (2009);RUC027WSF2024-223 each of which is incorporated by reference herein in their entirety.) The generation of induced pluripotent stem cells (iPSCs) is outlined below. As used herein, “hiPSCs” are human induced pluripotent stem cells, and “miPSCs” are murine induced pluripotent stem cells.

[0136] "Pluripotent stem cell characteristics" refer to characteristics of a cell that distinguish pluripotent stem cells from other cells. The ability to give rise to progeny that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm) is a pluripotent stem cell characteristic. Expression or non-expression of certain combinations of molecular markers are also pluripotent stem cell characteristics. For example, human pluripotent stem cells express at least several, and in some embodiments, all of the markers from the following non-limiting list: SSEA-3, SSEA- 4, TRA-1-60, TRA-1-81, TRA-2-49 / 6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rexl, and Nanog. Cell morphologies associated with pluripotent stem cells are also pluripotent stem cell characteristics. As described herein, cells do not need to pass through pluripotency to be reprogrammed into endodermal progenitor cells and / or hepatocytes.

[0137] As used herein, "multipotent" or "multipotent cell" refers to a cell type that can give rise to a limited number of other particular cell types. For example, induced multipotent cells are capable of forming endodermal cells. Additionally, multipotent blood stem cells can differentiate itself into several types of blood cells, including lymphocytes, monocytes, neutrophils, etc.

[0138] As used herein, the term "oligopotent" refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of either the lymphoid or myeloid lineages, respectively.

[0139] As used herein, the term "unipotent" means the ability of a cell to form a single cell type. For example, spermatogonial stem cells are only capable of forming sperm cells.

[0140] As used herein, the term "totipotent" means the ability of a cell to form an entire organism. For example, in mammals, only the zygote and the first cleavage stage blastomeres are totipotent.

[0141] As used herein, "non-pluripotent cells" refer to mammalian cells that are not pluripotent cells. Examples of such cells include differentiated cells as well as progenitorRUC027WSF2024-223 cells. Examples of differentiated cells include, but are not limited to, cells from a tissue selected from bone marrow, skin, skeletal muscle, fat tissue and peripheral blood. Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells. The starting cells employed for generating the induced multipotent cells, the endodermal progenitor cells, and the hepatocytes can be non-pluripotent cells.

[0142] Differentiated cells include, but are not limited to, multipotent cells, oligopotent cells, unipotent cells, progenitor cells, and terminally differentiated cells. In particular embodiments, a less potent cell is considered “differentiated” in reference to a more potent cell.

[0143] A "somatic cell" is a cell forming the body of an organism. Somatic cells include cells making up organs, skin, blood, bones and connective tissue in an organism, but not germ cells.

[0144] Cells can be from, for example, human or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, and non-human primates. In some embodiments, a cell is from an adult human or non-human mammal. In some embodiments, a cell is from a neonatal human, an adult human, or non-human mammal.

[0145] As used herein, the terms "subject" or "patient" refers to any animal, such as a domesticated animal, a zoo animal, or a human. The "subject" or "patient" can be a mammal like a dog, cat, bird, livestock, or a human. Specific examples of "subjects" and "patients" include, but are not limited to, individuals (particularly human) with a disease or disorder related to the liver, heart, lung, kidney, pancreas, brain, neural tissue, blood, bone, bone marrow, and the like.

[0146] Mammalian cells can be from humans or non-human mammals. Exemplary non-human mammals include, but are not limited to, mice, rats, cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, bovines, and non-human primates (e.g., chimpanzees, macaques, and apes).

[0147] “Hypoimmunogenic cells” or “hypoimmune cells” have a reduced immunological rejection response when transferred into an allogeneic host. They have been engineered to possess hypoimmunogenicity, e.g., by the reduction of HLA-I function and the expression or overexpression of a SIRPa engager molecule on the cell surface. They mayRUC027WSF2024-223 also include a reduced HLA-II function on the cell surface. These may be accomplished by modifications that reduce or knock out B2M expression, forced CD47 transgene expression, and, optionally, reduced or knock out CIITA expression. Thus, “hypoimmunogenic” refers to a significantly reduced or eliminated immune response when compared to the immune response of a parental (i.e. “wt”) cell prior to immunoengineering as outlined herein. In preferred embodimements, HIP cells do not give rise to an immune response. Assays for hypoimmune characteristics are provided herein.

[0148] “Hypoimmune pluripotent cells” or “HIP cells” are hypoimmune cells that are pluripotent. In some embodiments, HIP cells or HIP cell derivatives are therapeutic cells. In many cases, the HIP cells are immunologically silent and yet retain pluripotent capabilities.

[0149] “HIPO- cells” are HIP cells that are also ABO blood type O and Rh factor negative (O-). In some embodiments of the invention, therapeutic cells (pluripotent or not) are not necessarily O- but are suitable for administering to a blood type-compatible recipient. For instance, an A+ cell or hypoimmune cell is suitable for administering to A+ or AB+ recipients, but not B+, B-, 0+ or O- recipients. There are patients with A- or AB- that have anti-Rh antibodies and could mount an immune response against A+ cells or hypoimmune cells and those cells would be killed.

[0150] “Comprehensively immune protected cells” or “CIP cells” are engineered to possess hypoimmunogenicity (e.g., reduced HLA-I function and a SIRPa engager molecule expressed on the cell surface) as well as the expression of an Fc sequestration molecule. The Fc sequestration molecules on the CIP cell surface may be, e.g., CD16, CD32, CD64, or modified or truncated versions thereof. The CIP cells may also include reduced HLA-II function. The modifications may reduce or knock out B2M expression and, optionally, additionally reduce or knockout CIITA. CIP cells may further comprise one or more modifications that evade host immune killing. Examples include cell-surface molecules that evade polymorphonuclear cell (PMN) killing such as CD200R engageers (e.g. CD200) and CD99 ligand engagers (e.g. CD99). The latter transgenes are particularly important for the use of the engineered cells in xenotransplantation.

[0151] The terms “differentiated hypoimmune pluripotent cells,” “differentiated HIP cells,” “dHIP cells,” “differentiated HIPO-,” “dHIPO- cells,” “differentiated comprehensively immune protected cells” or “dCIP cells” herein refer to cells that have been engineered to possess hypoimmunogenicity and / or comprehensive immune protection but are differentiated. These cells may have been differentiated into a cell type for ultimateRUC027WSF2024-223 transplantation or xenotransplantation into subjects. Thus, for example, CIP cells can be differentiated into hepatocytes (“dCIP hepatocytes”), into beta-like pancreatic cells or islet cells or organoids (“dCIP beta cells”), into endothelial cells (“dCIP endothelial cells”), thyroid cells, or parathyroid cells etc. Paralell definitions apply to, e.g., “differentiated HIP / CD64 cells” and “differentiated HIPO- / CD64 cells.”

[0152] These cells may be used for transplantation or xenotransplantation into subjects. They may be incorporated into a cellular, tissue, or organ therapies. The therapeutic cells have decreased or eliminated analogs to HLA-I and HLA-II. For example, therapeutic pig cells, tissues, or organs have reduced or eliminated swine leukocyte antigens I and II (SLA-I and SLA-II, respectively). Likewise, the therapeutic cells will be ABO blood-type and Rh compatible with the recipient. Additionally, the cells may have cell-surface engagers to SIRPa, CD200R, and CD99 ligands. They may also have cell-surface Fc sequestration molecules.

[0153] The CD99 ligand engager has an extracellular domain that engages with CD99 ligands (e.g. PILRs and CD99) and can have a heterologous TMD domain, wherein the TMD is selected from the group consting of CD47, CD64, CD85f, CD349, CD284, CD261, CD172b, CD277, CD186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CDla, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CDl lb, CD107a, CD331, CD222, CD20, CD79a, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CDle, CDld, CD227, CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CDl la, CDl lc, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CDlb, CDlc, CD154, CD6, CD52, CD132, CD32, CD66b, CD171, CD191, CD197, CD185, CD131, CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361, CD147, CD309, CD135, CD292, CD103, CD130,RUC027WSF2024-223CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231, CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD158i, CD178, CD295, CD151, CD97, CD183, CD39, CD239, CD193, CD194, CD195, CD196, CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241, CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91, CDw210b, CD167a, CD3OOc, CD157, CD317, CD148, CD161, CD215, CD150, CDlld, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111, CD281, CD226, CD234, CD167b, CD3OOe, CD276, CD305, CD3OOg, CD3OOd, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158z, CD158f2, CD85j, CD3OOf, CD92, CD351, CD112, CD 100, CD270, CD101, CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD 114, CD 180, CD158d, CD273, CD290, CD244, CD 169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201, CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51, CD41, CD29, CD 18, CD61, and CD 104.

[0154] Cells of the invention that express a heterologous CD200R engager molecule may be referred to herein as “CD200R engager” or “CD200R-E” cells. For example, a “CIP- CD200R-E cell” herein is meant a CIP cell that expresses a CD200R-E molecule.

[0155] By “hypo-immunogenic pluripotent cell O-” “hypo-immunogenic pluripotent ORh-” cell or “HIPO-” cell herein is meant a HIP cell that is also ABO blood group O and Rhesus Factor Rh-. HIPO- cells may have been generated from O- cells, enzymatically modified to be O-, or genetically engineered to be O-. For example, a “HIPO- / CD200R-E cell” herein is meant a HIPO- cell that expresses a CD200R-E molecule.

[0156] The CD200R-E cells of the invention may be derived or differentiated from, for example, an induced pluripotent stem cell, an embryonic stem cell, a HIP cell, a HIPO-, or a non-pluripotent cell type.

[0157] By “HLA” or “human leukocyte antigen” complex is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. These cellsurface proteins that make up the HLA complex are responsible for the regulation of the immune response to antigens. In humans, there are two MHCs, class I and class II, “HLA-I” and “HLA-II”. HLA-I includes three proteins, HL A- A, HLA-B and HLA-C, which presentRUC027WSF2024-223 peptides from the inside of the cell, and antigens presented by the HLA-I complex attract killer T-cells (also known as CD8+ T-cells or cytotoxic T cells). The HLA-I proteins are associated with P-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 should be understood that the use of either “MHC” or “HLA” is not meant to be limiting, as it depends on whether the genes are from humans (HLA) or murine (MHC). Thus, as it relates to mammalian cells, these terms may be used interchangeably herein.

[0158] By “gene knock out” herein is meant a process that renders a particular gene inactive in the host cell in which it resides, resulting either in no protein of interest being produced or an inactive form. As will be appreciated by those in the art and further described below, this can be accomplished in a number of different ways, including removing nucleic acid sequences from a gene, or interrupting the sequence with other sequences, altering the reading frame, or altering the regulatory components of the nucleic acid. For example, all or part of a coding region of the gene of interest can be removed or replaced with “nonsense” sequences, all or part of a regulatory sequence such as a promoter can be removed or replaced, translation initiation sequences can be removed or replaced, etc.

[0159] By “gene knock in” herein is meant a process that adds a genetic function to a host cell. This causes increased levels of the encoded protein. As will be appreciated by those in the art, this can be accomplished in several ways, including adding one or more additional copies of the gene to the host cell or altering a regulatory component of the endogenous gene increasing expression of the protein is made. This may be accomplished by modifying the promoter, adding a different promoter, adding an enhancer, or modifying other gene expression sequences.

[0160] “P-2 microglobulin” or “P2M” or “B2M” protein refers to the human P2M protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000015.10 :44711487-44718159.

[0161] “CD47 protein” protein refers to the human CD47 protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000003.12: 108043094-108094200.RUC027WSF2024-223

[0162] “CIITA protein” protein refers to the human CIITA protein that has the amino acid and nucleic acid sequences shown below; the human gene has accession number NC_000016.10: 10866208-10941562.

[0163] By “wild type” in the context of a cell means a cell found in nature. However, in the context of a pluripotent stem cell, as used herein, it also means an iPSC that may contain nucleic acid changes resulting in pluripotency but did not undergo the gene editing procedures of the invention to achieve hypo-immunogenicity.

[0164] By “syngeneic” herein refers to the genetic similarity or identity of a host organism and a cellular transplant where there is immunological compatibility; e.g. no immune response is generated.

[0165] By “allogeneic” herein refers to the genetic dissimilarity of a host organism and a cellular transplant where an immune response is generated.

[0166] By “xenogeneic” herein refers to the transfer of living cells, tissues, or organs from a donor of one species to a recipient of a different species (for example, from a pig to a human).

[0167] By “B2M- / -“ herein is meant that a diploid cell has had the B2M gene inactivated in both chromosomes. As described herein, this can be done in a variety of ways.

[0168] By “CIITA- / -“ herein is meant that a diploid cell has had the CIITA gene inactivated in both chromosomes. As described herein, this can be done in a variety of ways.

[0169] By “CD47 tg” (standing for “transgene”) or “CD47+”) herein is meant that the host cell expresses CD47, in some cases by having at least one additional copy of the CD47 gene.

[0170] An "Oct polypeptide" refers to any of the naturally-occurring members of Octamer family of transcription factors, or variants thereof that maintain transcription factor activity, similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain. Exemplary Oct polypeptides include Oct-1, Oct-2, Oct-3 / 4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. Oct3 / 4 (referred to herein as "Oct4") contains the POU domain, a 150 amino acid sequence conserved among Pit-1, Oct-1, Oct-2, and uric-86. (See, Ryan, A. K. & Rosenfeld, M. G., Genes Dev. 11 : 1207-1225 (1997), incorporated herein by reference in its entirety.) In some embodiments, variants have at least 85%, 90%, or 95% amino acidRUC027WSF2024-223 sequence identity across their whole sequence compared to a naturally occurring Oct polypeptide family member such as to those listed above or such as listed in Genbank accession number NP-002692.2 (human Oct4) or NP-038661.1 (mouse Oct4). Oct polypeptides (e.g., Oct3 / 4 or Oct 4) can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. The Oct polypeptide(s) can be a pluripotency factor that can help induce multipotency in non-pluripotent cells.

[0171] A "Klf polypeptide" refers to any of the naturally-occurring members of the family of Kriippel-like factors (Klfs), zinc-finger proteins that contain amino acid sequences similar to those of the Drosophila embryonic pattern regulator Kriippel, or variants of the naturally-occurring members that maintain transcription factor activity similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain. (See, Dang, D. T., Pevsner, J. & Yang, V. W., Cell Biol. 32: 1103-1121 (2000), incorporated by reference herein in its entirety.) Exemplary Klf family members include, Klfl, Klf2, Klf3, Klf-4, Klf5, Klf6, KIH, Klf8, Klf9, KlflO, Klfl 1, Klfl2, Klfl3, Klfl4, Klfl5, Klfl 6, and Klfl 7. Klf2 and Klf-4 were found to be factors capable of generating iPS cells in mice, and related genes Klfl and Klf5 did as well, although with reduced efficiency. (See, Nakagawa, et al., Nature Biotechnology 26: 101-106 (2007), incorporated by reference herein in its entirety.) In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Klf polypeptide family member such as to those listed above or such as listed in Genbank accession number CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klf polypeptides (e.g., Klfl, Klf4, and Klf5) can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. The Klf polypeptide(s) can be a pluripotency factor. The expression of the Klf4 gene or polypeptide can help induce multipotency in a starting cell or a population of starting cells.

[0172] A "Myc polypeptide" refers to any of the naturally-occurring members of the Myc family. (See, e.g., Adhikary, S. & Eilers, M., Nat. Rev. Mol. Cell Biol. 6:635-645 (2005), incorporated by reference herein in its entirety.) It also includes variants that maintain similar transcription factor activity when compared to the closest related naturally occurringRUC027WSF2024-223 family member (i.e. within at least 50%, 80%, or 90% activity). It further includes polypeptides comprising at least the DNA-binding domain of a naturally occurring family member, and can further comprise a transcriptional activation domain. Exemplary Myc polypeptides include, e.g., c-Myc, N-Myc and L-Myc. In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Myc polypeptide family member, such as to those listed above or such as listed in Genbank accession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc) can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. The Myc polypeptide(s) can be a pluripotency factor.

[0173] A "Sox polypeptide" refers to any of the naturally-occurring members of the SRY-related HMG-box (Sox) transcription factors, characterized by the presence of the high- mobility group (HMG) domain, or variants thereof that maintain similar transcription factor activity when compared to the closest related naturally occurring family member i.e. within at least 50%, 80%, or 90% activity). It also includes polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain. (See, e.g., Dang, D. T. et al., Ini. J. Biochem. Cell Biol.32: 1103-1121 (2000), incorporated by reference herein in its entirety.) Exemplary Sox polypeptides include, e.g., Soxl, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, SoxlO, Soxl l, Soxl2, Soxl3, Soxl4, Soxl5, Soxl7, Soxl8, Sox-21, and Sox30. Soxl has been shown to yield iPS cells with a similar efficiency as Sox2, and genes Sox3, Soxl 5, and Soxl 8 have also been shown to generate iPS cells, although with somewhat less efficiency than Sox2. (See, Nakagawa, et al., Nature Biotechnology 26: 101-106 (2007), incorporated by reference herein in its entirety.) In some embodiments, variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Sox polypeptide family member such as to those listed above or such as listed in Genbank accession number CAA83435 (human Sox2). Sox polypeptides (e.g., Soxl, Sox2, Sox3, Soxl 5, or Soxl 8) can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. The Sox polypeptide(s) can be a pluripotency factor. As discussed herein, SOX2 proteins find particular use in the generation of iPSCs.

[0174] 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 specifiedRUC027WSF2024-223 percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0175] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0176] One example of an algorithm that is 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 analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0177] “Inhibitors,” “activators,” and “modulators” affect a function or expression of a biologically-relevant molecule. The term “modulator” includes both inhibitors and activators. They may be identified using in vitro and in vivo assays for expression or activity of a target molecule.

[0178] “Inhibitors” are agents that, e.g., inhibit expression or bind to target molecules or proteins. They may partially or totally block stimulation or have protease inhibitor activity. They may reduce, decrease, prevent, or delay activation, including inactivation, desensitizion, or down regulation of the activity of the described target protein. Modulators may be antagonists of the target molecule or protein.RUC027WSF2024-223

[0179] “Activators” are agents that, e.g., induce or activate the function or expression of a target molecule or protein. They may bind to, stimulate, increase, open, activate, or facilitate the target molecule activity. Activators may be agonists of the target molecule or protein.

[0180] “Homologs” are bioactive molecules that are similar to a reference molecule at the nucleotide sequence, peptide sequence, functional, or structural level. Homologs may include sequence derivatives that share a certain percent identity with the reference sequence. Thus, in one embodiment, homologous or derivative sequences share at least a 70 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least an 80 or 85 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least a 90 percent sequence identity. In a specific embodiment, homologous or derivative sequences share at least a 95 percent sequence identity. In a more specific embodiment, homologous or derivative sequences share at least a 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity. Homologous or derivative nucleic acid sequences may also be defined by their ability to remain bound to a reference nucleic acid sequence under high stringency hybridization conditions. Homologs having a structural or functional similarity to a reference molecule may be chemical derivatives of the reference molecule. Methods of detecting, generating, and screening for structural and functional homologs as well as derivatives are known in the art.

[0181] “Hybridization” generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), incorporated by reference herein in its entirety.

[0182] "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.RUC027WSF2024-223

[0183] "Stringent conditions" or "high stringency conditions", as defined herein, can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 Mm sodium phosphate buffer at Ph 6.5 with 750 Mm sodium chloride, 75 Mm sodium citrate at 42°C; or (3) overnight hybridization in a solution that employs 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 Mm sodium phosphate (Ph 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 pl / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with a 10 minute wash at 42°C in 0.2 x SSC (sodium chloride / sodium citrate) followed by a 10 minute high- stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.

[0184] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0185] The term “about” has it’s ordinary and customary meaning as understood by a person of ordinary skill in the relevant art. In some aspects, when referring to a numerical value, the term “about” may refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10 %, 11%, 12%, 13%, 14%, or a 15% deviation from the numerical value.

[0186] As used herein the term “modification” refers to an alteration that physically differentiates the modified molecule from the parent molecule. In one embodiment, an amino acid change in a CD200, anti-CD200R domain, CD47, HSVtk, EC-CD, or iCasp9 variant polypeptide prepared according to the methods described herein differentiates it from the corresponding parent that has not been modified according to the methods described herein, such as wild-type proteins, a naturally occurring mutant proteins or another engineered protein that does not include the modifications of such variant polypeptide. In another embodiment, a variant polypeptide includes one or more modifications that differentiates the function of the variant polypeptide from the unmodified polypeptide. For example, an amino acid change in a variant polypeptide affects its receptor binding profile. In otherRUC027WSF2024-223 embodiments, a variant polypeptide comprises substitution, deletion, or insertion modifications, or combinations thereof. In another embodiment, a variant polypeptide includes one or more modifications that increases its affinity for a receptor compared to the affinity of the unmodified polypeptide.

[0187] In one embodiment, a variant polypeptide includes one or more substitutions, insertions, or deletions relative to a corresponding native or parent sequence. In certain embodiments, a variant polypeptide includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31-40, 41 to 50, or 51 or more modifications.

[0188] By “episomal vector” herein is meant a genetic vector that can exist and replicate autonomously in the cytoplasm of a cell; e.g. it is not integrated into the genomic DNA of the host cell. A number of episomal vectors are known in the art and described below.

[0189] By “knock out” in the context of a gene means that the host cell harboring the knock out does not produce a functional protein product of the gene. As outlined herein, a knock out can result in a variety of ways, from removing all or part of the coding sequence, introducing frameshift mutations such that a functional protein is not produced (either truncated or nonsense sequence), removing or altering a regulatory component (e.g. a promoter) such that the gene is not transcribed, preventing translation through binding to mRNA, etc. Generally, the knock out is effected at the genomic DNA level, such that the cells’ offspring also carry the knock out permanently.

[0190] By “knock in” in the context of a gene means that the host cell harboring the knock in has more functional protein active in the cell. As outlined herein, a knock in can be done in a variety of ways, usually by the introduction of at least one copy of a transgene (tg) encoding the protein into the cell, although this can also be done by replacing regulatory components as well, for example by adding a constitutive promoter to the endogeneous gene. In general, knock in technologies result in the integration of the extra copy of the transgene into the host cell.VII. Cells of the Invention

[0191] The invention provides compositions and methodologies for transplanted cells to evade the innate immune system. The invention blocks target cell killing by PMNs, macrophages, and NK cells by engaging CD200R on the immune cells (Figure 1). In someRUC027WSF2024-223 additional aspects of the invention, the cells will be derived from induced pluripotent stem cells (IPSC), O- induced pluripotent stem cells (iPSCO-), embryonic stem cells (ESC), O- embryonic stem cells (ESCO-), hypoimmunogenic pluripotent (HIP) cells, hypoimmunogenic pluripotent O- (HIPO-) cells, or cells derived or differentiated therefrom. In other additional aspects, the transplanted cells will be pluripotent with enhanced CD 16, CD32, OR CD64 expression, or cells derived therefrom.A. Methodologies for Genetic Alterations

[0192] The invention includes methods of modifying nucleic acid sequences within cells or in cell-free conditions. Exemplary technologies include homologous recombination, knock-in, ZFNs (zinc finger nucleases), TALENs (transcription activator-like effector nucleases), CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9, and other site-specific nuclease technologies. These techniques enable double-strand DNA breaks at desired locus sites. These controlled double-strand breaks promote homologous recombination at the 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 a double-stranded break in the nucleic acid molecule. The double-strand break is repaired either by an error-prone non-homologous end-joining (NHEJ) or by homologous recombination (HR).

[0193] As will be appreciated by those in the art, a number of different techniques can be used to engineer the pluripotent cells of the invention. In general, these techniques can be used individually or in combination. For example, in the generation of the HIP cells, CRISPR may be used to reduce the expression of active B2M and / or CIITA protein in the engineered cells, with viral techniques (e.g. lentivirus) to knock in the CD47 functionality. Also, as will be appreciated by those in the art, although one embodiment sequentially utilizes a CRISPR step to knock out B2M, followed by a CRISPR step to knock out CIITA with a final step of a lentivirus to knock in the CD47 functionality, these genes can be manipulated in different orders using different technologies.

[0194] As is discussed more fully below, transient expression of reprogramming genes is generally done to generate / induce pluripotent stem cells. a. CRISPR Technologies

[0195] In one embodiment, the cells are manipulated using clustered regularly interspaced short palindromic repeats) / Cas (“CRISPR”) technologies as is known in the art. CRISPR can be used to generate the starting iPSCs or to generate the HIP cells from theRUC027WSF2024-223 iPSCs. There are a large number of techniques based on CRISPR, see for example Doudna and Charpentier, Science doi: 10.1126 / science.1258096, hereby incorporated by reference. CRISPR techniques and kits are sold commercially. b. TALEN Technologies

[0196] In some embodiments, the HIP cells of the invention are made using Transcription Activator-Like Effector Nucleases (TALEN) methodologies. TALEN are restriction enzymes combined with a nuclease that can be engineered to bind to and cut practically any desired DNA sequence. TALEN kits are sold commercially. c. Zinc Finger Technologies

[0197] In one embodiment, the cells are manipulated using Zn finger nuclease technologies. Zn finger nucleases are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences and this enables zinc-finger nucleases to target unique sequences within complex genomes. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms, similar to CRISPR and TALENs. d. Viral Based Technologies

[0198] There are a wide variety of viral techniques that can be used to generate the HIP cells of the invention (as well as for the original generation of the iPCSs), including, but not limited to, the use of retroviral vectors, lentiviral vectors, adenovirus vectors and Sendai viral vectors. Episomal vectors used in the generation of iPSCs are described below. e. Down regulation of genes using interfering RNA

[0199] In other embodiments, genes that encode proteins used in HLA molecules are downregulated by RNAi technologies. RNA interference (RNAi) is a process where RNA molecules inhibit gene expression often by causing specific mRNA molecules to degrade. Two types of RNA molecules - microRNA (miRNA) and small interfering RNA (siRNA) - are central to RNA interference. They bind to the target mRNA molecules and either increase or decrease their activity. RNAi helps cells defend against parasitic nucleic acids such as those from viruses and transposons. RNAi also influences development.

[0200] sdRNA molecules are a class of asymmetric siRNAs comprising a guide (antisense) strand of 19-21 bases. They contain a 5’ phosphate, 2’0me or 2’F modified pyrimidines, and six phosphotioates at the 3’ positions. They also contain a sense strandRUC027WSF2024-223 containing 3’ conjugated sterol moi eties, 2 phospotioates at the 3’ position, and 2’0me modified pyrimidines. Both strands contain 2’ Ome purines with continuous stretches of unmodified purines not exceeding a length of 3. sdRNA is disclosed in U.S. Patent No. 8,796,443, incorporated herein by reference in its entirety.

[0201] For all of these technologies, well known recombinant techniques are used, to generate recombinant nucleic acids as outlined herein. In certain embodiments, the recombinant nucleic acids (either than encode a desired polypeptide, e.g. CD47, or disruption sequences) may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory nucleotide sequences will generally be appropriate for the host cell and subject to be treated. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are also contemplated. The promoters may be either naturally occurring promoters or hybrid promoters that combine elements of more than one promoter. An expression construct may be present in a cell on an episome such as a plasmid. Alternatively, the expression construct may be inserted in a chromosome. In a specific embodiment, the expression vector includes a selectable marker gene to allow the selection of transformed host cells. Certain embodiments include an expression vector comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory sequence. Regulatory sequence for use herein include promoters, enhancers, and other expression control elements. In certain embodiments, an expression vector is designed for the choice of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the vector's copy number, the ability to control that copy number, or the expression of any other protein encoded by the vector, such as antibiotic markers.

[0202] Examples of suitable mammalian promoters include, for example, promoters from the following genes: ubiquitin / S27a promoter of the hamster (WO 97 / 15664), Simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, the long terminal repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus Long Terminal repeat region, and the early promoter of human Cytomegalovirus (CMV). ExamplesRUC027WSF2024-223 of other heterologous mammalian promoters are the actin, immunoglobulin or heat shock promoter(s).

[0203] In additional embodiments, promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). In further embodiments, heterologous mammalian promoters are used. Examples include the actin promoter, an immunoglobulin promoter, and heat-shock promoters. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers et al., Nature 273: 113-120 (1978). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. Greenaway, P. J. et al., Gene 18: 355-360 (1982). The foregoing references are incorporated by reference in their entirety.B. Generation of Pluripotent Cells

[0204] The generation of mouse and human pluripotent stem cells (generally referred to as iPSCs; miPSCs for murine cells or hiPSCs for human cells) is generally known in the art. As will be appreciated by those in the art, there are a variety of different methods for the generation of iPCSs. The original induction was done from mouse embryonic or adult fibroblasts using the viral introduction of four transcription factors, Oct3 / 4, Sox2, c-Myc and Klf4; see Takahashi and Yamanaka Cell 126:663-676 (2006), hereby incorporated by reference in its entirety and specifically for the techniques outlined therein. Since then, a number of methods have been developed; see Seki et al., World J. Stem Cells 7(1): 116-125 (2015) for a review, and Lakshmipathy and Vermuri, editors, Methods in Molecular Biology: Pluripotent Stem Cells, Methods and Protocols, Springer 2013, both of which are hereby expressly incorporated by reference in their entirety, and in particular for the methods for generating hiPSCs (see for example Chapter 3 of the latter reference).

[0205] Generally, iPSCs are generated by the transient expression of one or more “reprogramming factors” in the host cell, usually introduced using episomal vectors. Under these conditions, small amounts of the cells are induced to become iPSCs (in general, the efficiency of this step is low, as no selection markers are used). Once the cells are “reprogrammed”, and become pluripotent, they lose the episomal vector(s) and produce the factors using the endogeneous genes. This loss of the episomal vector(s) results in cells that are called “zero footprint” cells. This is desirable as the fewer genetic modificationsRUC027WSF2024-223(particularly in the genome of the host cell), the better. Thus, it is preferred that the resulting hiPSCs have no permanent genetic modifications.

[0206] As is also appreciated by those of skill in the art, the number of reprogramming factors that can be used or are used can vary. Commonly, when fewer reprogramming factors are used, the efficiency of the transformation of the cells to a pluripotent state goes down, as well as the “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.

[0207] In some embodiments, a single reprogramming factor, OCT4, is used. In other embodiments, two reprogramming factors, OCT4 and KLF4, are used. In other embodiments, three reprogramming factors, OCT4, KLF4 and SOX2, are used. In other embodiments, four reprogramming factors, OCT4, KLF4, SOX2 and c-Myc, are used. In other embodiments, 5, 6 or 7 reprogramming factors can be used selected from SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28, and SV40L T antigen.

[0208] In general, these reprogramming factor genes are provided on episomal vectors such as are known in the art and commercially available. For example, Therm oFisher / Invitrogen sell a sendai virus reprogramming kit for zero footprint generation of hiPSCs, see catalog number A34546. ThermoFisher also sells EBNA-based systems as well, see catalog number A14703.

[0209] In addition, there are a number of commercially available hiPSC lines available; see, e.g., the Gibco® Episomal hiPSC line, KI 8945, which is a zero footprint, viral -integration-free human iPSC cell line (see also Burridge et al, 2011, supra).

[0210] In general, as is known in the art, iPSCs are made from non-pluripotent cells such as CD34+ cord blood cells, fibroblasts, etc., by transiently expressing the reprogramming factors as described herein.

[0211] For example, successful iPSCs were also generated using only Oct3 / 4, Sox2 and Klf4, while omitting the C-Myc, although with reduced reprogramming efficiency.

[0212] In general, iPSCs are characterized by the expression of certain factors that include KLF4, Nanog, OCT4, SOX2, ESRRB, TBX3, c-Myc and TCL1. New or increased expression of these factors for purposes of the invention may be via induction or modulation of an endogenous locus or from expression from a transgene.RUC027WSF2024-223

[0213] For example, murine iPSCs can be generated using the methods of Diecke et al, Sci Rep. 2015, Jan. 28;5:8081 (doi:10.1038 / srep08081), hereby incorporated by reference in its entirety and specifically for the methods and reagents for the generation of the miPSCs. See also, e.g., Burridge et al. , PLoS One, 2011 6(4): 18293, hereby incorporated by reference in its entirety and specifically for the methods outlined therein.

[0214] In some cases, the pluripotency of the cells is measured or confirmed as outlined herein, for example by assaying for reprogramming factors or by conducting differentiation reactions as outlined herein and in the Examples.C. Generation of Hypo-Immunogenic Pluripotent (HIP) Cells

[0215] Generating HIP cells from pluripotent cells is done with as few as three genetic changes, resulting in minimal disruption of cellular activity but conferring immunosilencing to the cells.

[0216] As discussed herein, one embodiment utilizes a reduction or elimination of gene activity or gene products in an MHC I and / or II complex (HLA I and II when the cells are human). This can be done by altering genes encoding one or more components of the MHC or HLA complex. In one embodiment, the coding region or regulatory sequences of the gene are disrupted using CRISPR. In another embodiment, gene translation is reduced using interfering RNA technologies. The third change is the introduction or upregulation of a gene whose product reduces susceptibility to macrophage phagocytosis. In one example, CD47 is introduced on a transgene or an endogenous CD47 is upregulated to express the CD47 above a threshold required to resist macrophages and / or NK cell killing. Alternatively, other natural or engineered SIRPa engagers are introduced into the cell.

[0217] In some cases, where CRISPR is being used for the genetic modifications, hiPSC cells that contain a Cas9 construct that enable high efficiency editing of the cell line can be used; see, e.g., the Human Episomal Cas9 iPSC cell line, A33124, from Life Technologies.1. HL A-I Reduction

[0218] The HIP cells of the invention include a reduction in MHC I function (HLA I when the cells are derived from human cells).

[0219] As will be appreciated by those in the art, the reduction in function can be accomplished in a number of ways, including removing nucleic acid sequences from a gene, interrupting the sequence with other sequences, or altering the regulatory components of theRUC027WSF2024-223 nucleic acid. For example, all or part of a coding region of the gene of interest can be removed or replaced with “nonsense” sequences, frameshift mutations can be made, all or part of a regulatory sequence such as a promoter can be removed or replaced, translation initiation sequences can be removed or replaced, etc.

[0220] As will be appreciated by those in the art, the successful reduction of the MHC I function (HLA I when the cells are derived from human cells) in the pluripotent cells can be measured using techniques known in the art and as described below; for example, FACS techniques using labeled antibodies that bind the HLA complex; for example, using commercially available HLA-A,B,C antibodies that bind to the the alpha chain of the human major histocompatibility HLA Class I antigens. a. B2M Alteration

[0221] In one embodiment, the reduction in HLA-I activity is done by disrupting the expression of the P-2 microglobulin gene in the pluripotent stem cell, the human sequence of which is disclosed herein. This alteration is generally referred to herein as a gene “knock out”, and in the HIP cells of the invention it is done on both alleles in the host cell. Generally the techniques to do both disruptions is the same.

[0222] A particularly useful embodiment uses CRISPR technology to disrupt the gene. In some cases, CRISPR technology is used to introduce small deletions / insertions into the coding region of the gene, such that no functional protein is produced, often the result of frameshift mutations that result in the generation of stop codons such that truncated, nonfunctional proteins are made.

[0223] Accordingly, a useful technique is to use CRISPR sequences designed to target the coding sequence of the B2M gene in mouse or the B2M gene in human. After gene editing, the transfected iPSC cultures are dissociated to single cells. Single cells are expanded to full-size colonies and tested for CRISPR edit by screening for presence of aberrant sequence from the CRISPR cleavage site. Clones with deletions in both alleles are picked. Such clones did not express B2M as demonstrated by PCR and did not express HLA-I as demonstrated by FACS analysis (see examples 1 and 6, for example).

[0224] Assays to test whether the B2M gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cells lysates probed with antibodies to the B2M protein. In another embodiment, reverse transcriptase polymerase chain reactions (rt-PCR) confirms the presence of the inactivating alteration.RUC027WSF2024-223

[0225] In addition, the cells can be tested to confirm that the HLA I complex is not expressed on the cell surface. This may be assayed by FACS analysis using antibodies to one or more HLA cell surface components as discussed above.

[0226] It is noteworthy that others have had poor results when trying to silence the B2M genes at both alleles. See, e.g. Gornalusse et al., Nature Biotech.Doi / 10.1038 / nbt.3860).2. HLA-II Reduction

[0227] In addition to a reduction in HLA I, the HIP cells of the invention also lack MHC II function (HLA II when the cells are derived from human cells).

[0228] As will be appreciated by those in the art, the reduction in function can be accomplished in a number of ways, including removing nucleic acid sequences from a gene, adding nucleic acid sequences to a gene, disrupting the reading frame, interrupting the sequence with other sequences, or altering the regulatory components of the nucleic acid. In one embodiment, all or part of a coding region of the gene of interest can be removed or replaced with “nonsense” sequences. In another embodiment, regulatory sequences such as a promoter can be removed or replaced, translation initiation sequences can be removed or replaced, etc.

[0229] The successful reduction of the MHC II function (HLA II when the cells are derived from human cells) in the pluripotent cells or their derivatives can be measured using techniques known in the art such as Western blotting using antibodies to the protein, FACS techniques, rt-PCR techniques, etc. a. CIITA Alteration

[0230] In one embodiment, the reduction in HLA-II activity is done by disrupting the expression of the CIITA gene in the pluripotent stem cell, the human sequence of which is shown herein. This alteration is generally referred to herein as a gene “knock out”, and in the HIP cells of the invention it is done on both alleles in the host cell.

[0231] Assays to test whether the CIITA gene has been inactivated are known and described herein. In one embodiment, the assay is a Western blot of cells lysates probed with antibodies to the CIITA protein. In another embodiment, reverse transcriptase polymerase chain reactions (rt-PCR) confirms the presence of the inactivating alteration.RUC027WSF2024-223

[0232] In addition, the cells can be tested to confirm that the HLA II complex is not expressed on the cell surface. Again, this assay is done as is known in the art. Exemplary analyses include Western Blots or FACS analysis using commercial antibodies that bind to human HLA Class II HLA-DR, DP and most DQ antigens as outlined below.

[0233] A particularly useful embodiment uses CRISPR technology to disrupt the CIITA gene. CRISPRs were designed to target the coding sequence of the Ciita gene in mouse or the CIITA gene in human, an essential transcription factor for all MHC II molecules. After gene editing, the transfected iPSC cultures were dissociated into single cells. They were expanded to full-size colonies and tested for successful CRISPR editing by screening for the presence of an aberrant sequence from the CRISPR cleavage site. Clones with deletions did not express CIITA as determined by PCR and did not express MHC II / HLA-II as determined by FACS analysis.3. Phagocytosis Reduction

[0234] In addition to the reduction of HLA I and II (or MHC I and II), generally using B2M and CIITA knock-outs, the HIP cells of the invention have a reduced susceptibility to macrophage phagocytosis and NK cell killing. The resulting HIP cells “escape” the immune macrophage and innate pathways due to one or more CD47 transgenes. a. CD47 Increase

[0235] In some embodiments, reduced macrophage phagocytosis and NK cell killing susceptibility results from increased CD47 on the HIP cell surface. This is done in several ways as will be appreciated by those in the art using “knock in” or transgenic technologies. In some cases, increased CD47 expression results from one or more CD47 transgene.

[0236] Accordingly, in some embodiments, one or more copies of a CD47 gene is added to the HIP cells under control of an inducible or constitutive promoter, with the latter being preferred. In some embodiments, a lentiviral construct is employed as described herein or known in the art. CD47 genes may integrate into the genome of the host cell under the control of a suitable promoter as is known in the art.

[0237] The HIP cell lines were generated from B2M- / - CIITA- / - iPSCs. Cells containing lentivirus vectors expressing CD47 were selected using a Blasticidin marker. The CD47 gene sequence was synthesized and the DNA was cloned into the plasmid Lentivirus pLenti6 / V5 with a blasticidin resistance (Thermo Fisher Scientific, Waltham, MA)RUC027WSF2024-223

[0238] In some embodiments, the expression of the CD47 gene can be increased by altering the regulatory sequences of the endogenous CD47 gene, for example, by exchanging the endogenous promoter for a constitutive promoter or for a different inducible promoter. This can generally be done using known techniques such as CRISPR.

[0239] Once altered, the presence of sufficient CD47 expression can be assayed using known techniques such as those described in the Examples, such as Western blots, ELISA assays or FACS assays using anti-CD47 antibodies. In general, “sufficiency” in this context means an increase in the expression of CD47 on the HIP cell surface that silences NK cell killing. The natural expression levels on cells is too low to protect them from NK cell lysis once their MHC I is removed.4. Suicide Genes

[0240] In some embodiments, the invention provides hypoimmunogenic pluripotent cells that comprise a "suicide gene" or “suicide switch”. These are incorporated to function as a "safety switch" that can cause the death of the hypoimmunogenic pluripotent cells should they grow and divide in an undesired manner. The "suicide gene" ablation approach includes a suicide gene in a gene transfer vector encoding a protein that results in cell killing only when activated by a specific compound. A suicide gene may encode an enzyme that selectively converts a nontoxic compound into highly toxic metabolites. The result is specifically eliminating cells expressing the enzyme. In some embodiments, the suicide gene is the herpesvirus thymidine kinase (HSV-tk) gene and the trigger is ganciclovir. In other embodiments, the suicide gene is the Escherichia coli cytosine deaminase (EC-CD) gene and the trigger is 5 -fluorocytosine (5 -FC) (Barese et al., Mol. Therap. 20(10): 1932-1943 (2012), Xu et al., Cell Res. 8:73-8 (1998), both incorporated herein by reference in their entirety.)

[0241] In other embodiments, the suicide gene is an inducible Caspase protein. An inducible Caspase protein comprises at least a portion of a Caspase protein capable of inducing apoptosis. In one embodiment, the portion of the Caspase protein is exemplified in SEQ ID NO: 13. In preferred embodiments, the inducible Caspase protein is iCasp9. It comprises the sequence of the human FK506-binding protein, FKBP12, with an F36V mutation, connected through a series of amino acids to the gene encoding human caspase 9. FKBP12-F36V binds with high affinity to a small-molecule dimerizing agent, API 903. Thus, the suicide function of iCasp9 in the instant invention is triggered by the administration of a chemical inducer of dimerization (CID). In some embodiments, the CID is the small molecule drug API 903. Dimerization causes the rapid induction of apoptosis. (SeeRUC027WSF2024-223WO201 1146862; Stasi et al, N Engl. J. A et / 365;18 (2011); Tey et al., Biol. BloodMarrow Transplant. 13:913-924 (2007), each of which are incorporated by reference herein in their entirety.)5. CD 16, CD32, or CD64 Expression

[0242] In some aspects of the invention, the cells have a reduced susceptibility to ADCC or CDC resulting from increased expression of CD 16, CD32, CD64, or truncated or mutated CD 16, CD32, or CD64. The resulting cells sequester antibodies due to the increased expression. In one embodiment, the cells comprise one or more CD 16, CD32, or CD64 transgenes.

[0243] The antibody sequestering molecule can have a CD64 extracellular domain and can have a heterologous TMD domain, wherein the TMD is selected from the group consting of CD99, CD47, CD85f, CD349, CD284, CD261, CD 172b, CD277, CD 186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CDla, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD8b, CD37, CDl lb, CD107a, CD331, CD222, CD20, CD79a, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD 119, CD 116, CD46, CDle, CD Id, CD227, CD44, CD62P, CD 104, CD43, CD 140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CDl la, CDl lc, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CD lb, CDlc, CD 154, CD6, CD52, CD 132, CD32, CD66b, CD 171, CD 191, CD 197, CD 185, CD131, CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361, CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231, CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD158i, CD178, CD295, CD151, CD97, CD183, CD39, CD239, CD193, CD194, CD195, CD196, CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125,RUC027WSF2024-223CD241, CD269, CD202b, CD87, CD 164, CD 136, CD 137, CD249, CD69, CD91, CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161, CD215, CD150, CDl ld, CD218a, CD210, CD 166, CD 162, CD213a2, CD242, CD 158g, CD158h, CD279, CD111, CD281, CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158z, CD158f2, CD85j, CD300f, CD92, CD351, CD 112, CD 100, CD270, CD 101, CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD 114, CD 180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201, CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51, CD41, CD29, CD 18, CD61, and CD 104. a. Increased CD 16, CD32, or CD64

[0244] In some aspects, reduced ADCC or CDC susceptibility results from increased CD 16, CD32, or CD64, on the cell surface. In some embodiments, the reduced ADCC or CDC results from a truncated version, for example, CD64.140a (SEQ ID NO:24) as described herein. This is done in several ways as will be appreciated by those in the art using “knock in” or transgenic technologies. In some cases, increased CD 16, CD32, or CD64 expression results from one or more transgenes.

[0245] Accordingly, in some embodiments, one or more copies of a CD 16, CD32, or CD64 gene is added to the cells under control of an inducible or constitutive promoter, with the latter being preferred. In some embodiments, a lentiviral construct is employed as described herein or known in the art. The genes may integrate into the genome of the host cell under the control of a suitable promoter as is known in the art.

[0246] Cells containing lentivirus vectors expressing CD 16, CD32, or CD64 are selected using a Blasticidin marker. The gene sequence is synthesized and the DNA may be cloned, for instance, into the plasmid Lentivirus pLenti6 / V5 with a blasticidin resistance (Thermo Fisher Scientific, Waltham, MA)

[0247] In some embodiments, the expression of the gene can be increased by altering the regulatory sequences of the endogenous CD 16, CD32, or CD64 gene, for example, by exchanging the endogenous promoter for a constitutive promoter or for a different inducible promoter. This can generally be done using known techniques such as CRISPR.RUC027WSF2024-223

[0248] Once altered, the presence of sufficient expression can be assayed using known techniques such as those described in the Examples, such as Western blots, ELISA assays or FACS assays using anti-CD16, CD32, or CD64 antibodies. In general, “sufficiency” in this context means an increase in expression the cell surface that sequesters antibodies and inhibis ADCC or CDC.6. Assays for HIP Phenotypes and Retention of Pluripotency

[0249] Once the HIP cells have been generated, they may be assayed for their hypoimmunogenicity and / or retention of pluripotency as is generally described herein and in the examples.

[0250] For example, hypo-immunogenicity are assayed using a number of techniques One exemplary technique includes transplantation into allogeneic hosts and monitoring for HIP cell growth (e.g. teratomas) that escape the host immune system. HIP derivatives are transduced to express luciferase and can then followed using bioluminescence imaging.Similarly, the T cell and / or B cell response of the host animal to the HIP cells are tested to confirm that the HIP cells do not cause an immune reaction in the host animal. T cell function is assessed by Elispot, Elisa, FACS, PCR, or mass cytometry (CYTOF). B cell response or antibody response is assessed using FACS or luminex. Additionally, or alternatively, the cells may be assayed for their ability to avoid innate immune responses, e.g. NK cell killing. NK cell lytolytic activity is assessed in vitro or in vivo using techniques known in the art.

[0251] Similarly, the retention of pluripotency is tested in a number of ways. In one embodiment, pluripotency is assayed by the expression of certain pluripotency-specific factors as generally described herein. Additionally or alternatively, the HIP cells are differentiated into one or more cell types as an indication of pluripotency.D. Generation of HIPO- with CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecules

[0252] In some aspects of the invention, the HIP cells generated as above will already be HIPO- cells because the process will have started with pluripotent cells having an O- blood type.

[0253] Other aspects of the invention involve the enzymatic conversion of A and B antigens. In preferred aspects, the B antigen is converted to O using an enzyme. In more preferred aspects, the enzyme is an a -galactosidase. This enzyme eliminates the terminal galactose residue of the B antigen. Other aspects of the invention involve the enzymaticRUC027WSF2024-223 conversion of A antigen to O. In preferred aspects, the A antigen is converted to O using an a-N-acetylgalactosaminidase. Enzymatic conversion is discussed, e.g., in Olsson et al., Transfusion Clinique et Biologique 11 :33-39 (2004); U.S. Pat. Nos. 4,427,777, 5,606,042, 5,633,130, 5,731,426, 6,184,017, 4, 609,627, and 5,606,042; and Int’l Pub. No. WO9923210, each of which are incorporated by reference herein in their entirety.

[0254] Other embodiments of the invention involve genetically engineering the cells by knocking out the ABO gene Exon 7 or silencing the SLC14A1 (JK) gene. Other embodiments of the invention involve knocking out the C and E antigens of the Rh blood group system (RH), K in the Kell system (KEL), Fya and Fy3 in the Duffy system (FY), Jkb in the Kidd system (JK), or U and S in the MNS blood group system. Any knockout methodology known in the art or described herein, such as CRISPR, talens, or homologous recombination, may be employed.E. Preferred Embodiments of the Invention

[0255] The cells of the invention that express one or more CD200R engagers, CD99 ligand engagers, SIRPa engagers, and / or Fc sequestration molecules may be used to treat, for example, Type 1 diabetes, type 2 diabetes, monogenic diabetes, cardiac diseases, neurological diseases, cancer, blindness, vascular diseases, and others that respond to regenerative medicine therapies. In particular, the invention contemplates using the cells for differentiation into any cell type. Particular aspects are described below:

[0256] In one aspect, the cells of the present invention comprise a nucleic acid encoding a chimeric antigen receptor (CAR). The CAR can comprise an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In other aspects, the CAR cell is derived from a HIP cell, a HIPO- cell, or overexpresses CD 16, CD32, or CD64.

[0257] In some embodiments, the CAR’s extracellular domain binds to an antigen selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CS1, CD171, BCMA, MUC16, ROR1, GPC3, WT1, CD30, CD33, CLL-1, CD70, LILRB4, Siglec-6, NPM1 neoepitope, FLT3, CD138, CD229, SLAMF7 / CS1, APRIL, GPRC5D, CD126, and TNF-NTF, GD2, HER2, MUC1, B7H3, EGFR, CEA, MSLN, NKG2D, CLDN18.2, EGFRvIII, CD44v6, CLEC14A, and FAP. In certain embodiments, the extracellular domain comprises a single chain variable fragment (scFv). In some embodiments, the transmembrane domain comprises CD3(^, CD4, CD8a, CD28, 4-1BB, 0X40, ICOS, CTLA-4,RUC027WSF2024-223PD-1, LAG-3, and BTLA. In certain embodiments, the intracellular signaling domain comprises CD3< CD28, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, and BTLA.

[0258] In certain embodiments, the CAR comprises an anti-CD19 scFv domain, a CD28 transmembrane domain, and a CD3 zeta signaling intracellular domain. In some embodiments, the CAR comprises anti-CD19 scFv domain, a CD28 transmembrane domain, a 4-1BB signaling intracellular domain, and a CD3 zeta signaling intracellular domain.

[0259] In another aspect of the invention, provided is an isolated CAR-T overexpressing cell CD 16, CD32, or CD64 produced by in vitro differentiation of any one of the cells described herein. In some embodiments, the cell is a cytotoxic hypoimmune CAR-T cell.

[0260] In various embodiments, the in vitro differentiation comprises culturing the cell carrying a CAR construct in a culture media comprising one or more growth factors or cytokines selected from the group consisting of bFGF, EPO, Flt3L, IGF, IL-3, IL-6, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture media further comprises one or more selected from the group consisting of a BMP activator, a GSK3 inhibitor, a ROCK inhibitor, a TGFP receptor / ALK inhibitor, and a NOTCH activator.

[0261] In particular embodiments, the isolated CAR-T cell of the invention produced by in vitro differentiation is used as a treatment of cancer.

[0262] In another aspect of the invention, provided is a method of treating a patient with cancer by administering a composition comprising a therapeutically effective amount of any of the isolated CAR-T cells described herein in comnbination with a CD200R engagement strategy. In some embodiments, the composition further comprises a therapeutically effective carrier.

[0263] In some embodiments, the administration step comprises intravenous administration, subcutaneous administration, intranodal administration, intratumoral administration, intrathecal administration, intrapleural administration, and intraperitoneal administration. In certain instances, the administration further comprises a bolus or by continuous perfusion.

[0264] In some embodiments, the cancer is a blood cancer selected from the group consisting of leukemia, lymphoma, and myeloma. In various embodiments, the cancer is a solid tumor cancer or a liquid tumor cancer.RUC027WSF2024-223

[0265] In another aspect, the present invention provides a method of making any one of the isolated CAR-T derived from a pluripotent cell including a HIP cell, a HIPO- cell, or one that overexpresses CD 16, CD32, or CD64 as described herein. The method includes in vitro differentiating of any one of the cells of the invention wherein in vitro differentiating comprises culturing them in a culture media comprising one or more growth factors or cytokines selected from the group consisting of bFGF, EPO, Flt3L, IGF, IL-2, IL-3, IL-6, IL- 7, IL-15, GM-CSF, SCF, and VEGF. In some embodiments, the culture media further comprises one or more selected from the group consisting of a BMP activator, a GSK3 inhibitor, a ROCK inhibitor, a TGFP receptor / ALK inhibitor, and a NOTCH activator.

[0266] In some aspects, the in vitro differentiating comprises culturing the pluripotent cells on feeder cells. In various embodiments, the in vitro differentiating comprises culturing in simulated microgravity. In certain instances, the culturing in simulated microgravity is for at least 72 hours.

[0267] In some aspects, provided herein is an isolated, therapeutic cardiac cell that is administered with a strategy for reducing PMN killing. In other aspects, provided herein is a method of treating a patient suffering from a heart condition or disease with a therapeutic cardiac cell and a strategy for reducing PMN killing. The method comprises administering a composition comprising a therapeutically effective amount of a population of any one of the isolated cardiac cells derived from cells of the invention as described herein in combination with a PMN inhibiting strategy. In some aspects, a PMN cell receptor is targeted. In other aspects, the cardiac cell has an elevated level of CD200 or CD99 expression. In other aspects, the cardiac cell has an Fc sequestration molecule. The composition further comprises a therapeutically effective carrier.

[0268] In some embodiments, the administration comprises implantation into the patient’s heart tissue, intravenous injection, intraarterial injection, intracoronary injection, intramuscular injection, intraperitoneal injection, intramyocardial injection, trans-endocardial injection, trans-epicardial injection, or infusion.

[0269] In some embodiments, the heart condition or disease is selected from the group consisting of pediatric cardiomyopathy, age-related cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, chronic ischemic cardiomyopathy, peripartum cardiomyopathy, inflammatory cardiomyopathy, other cardiomyopathy, myocarditis, myocardial ischemic reperfusion injury, ventricular dysfunction, heart failure, congestive heart failure, coronary artery disease, end stage heartRUC027WSF2024-223 disease, atherosclerosis, ischemia, hypertension, restenosis, angina pectoris, rheumatic heart, arterial inflammation, or cardiovascular disease.

[0270] In some aspects, provided herein is a method of using a population of hypoimmune cardiac cells from a population of HIP, HIPO-, or HIPO- cells with CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecules by in vitro differentiation, wherein endogenous P-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated and a SIRPa engager like CD47 is overexpressed in the HIPO-cells. The method comprises: (a) culturing a population of HIPO-cells in a culture medium comprising a GSK inhibitor; (b) culturing the population of HIPO-cells in a culture medium comprising a WNT antagonist to produce a population of pre-cardiac cells; and (c) culturing the population of pre-cardiac cells in a culture medium comprising insulin to produce a population of hypoimmune cardiac cells.

[0271] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 2 pM to about 10 pM. In some embodiments, the WNT antagonist is IWR1, a derivative thereof, or a variant thereof. In some instances, the WNT antagonist is at a concentration ranging from about 2 pM to about 10 pM.

[0272] Some aspects of the invention utilize a therapeutic endothelial cell in combination with a CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecules. In some aspects, the endothelia cell is derived from a HIP cell, a HIPO- cell, or overexpresses CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecules. In other aspects, the therapeutic endothelial cell of the invention is selected from the group consisting of a capillary endothelial cell, vascular endothelial cell, aortic endothelial cell, brain endothelial cell, and renal endothelial cell.

[0273] In some aspects, provided herein is a method of treating a patient suffering from a vascular condition or disease. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of a population of isolated, engineered endothelial cells of the invention in combination with a CD200R engagement, CD99 ligand engagement, and / or Fc sequestration engagement strategies. In some embodiments, the composition further comprises a therapeutically effective carrier or excipient. In some embodiments, the administration comprises implantation into the patient’s heart tissue, intravenous injection, intraarterial injection, intracoronary injection,RUC027WSF2024-223 intramuscular injection, intraperitoneal injection, intramyocardial injection, trans-endocardial injection, trans-epicardial injection, or infusion.

[0274] In some embodiments, the vascular condition or disease is selected from the group consisting of, vascular injury, cardiovascular disease, vascular disease, ischemic disease, myocardial infarction, congestive heart failure, hypertension, ischemic tissue injury, limb ischemia, stroke, neuropathy, and cerebrovascular disease.

[0275] In some aspects, provided herein is a method of producing a population of hypoimmune endothelial cells from a population of HIP, HIPO-, iPSC, iPSCO-, ESC, or ESCO- cells, including those that overexpress CD 16, CD32, or CD64, or derivatives thereof. The method comprises: (a) culturing a population of HIPO-cells in a first culture medium comprising a GSK inhibitor; (b) culturing the population of HIPO-cells in a second culture 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 culture medium comprising a ROCK inhibitor and an ALK inhibitor to produce a population of hypoimmune endothelial cells.

[0276] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 1 pM to about 10 pM. In some embodiments, the ROCK inhibitor is Y- 27632, a derivative thereof, or a variant thereof. In some instances, the ROCK inhibitor is at a concentration ranging from about 1 pM to about 20 pM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 0.5 pM to about 10 pM.

[0277] In some embodiments, the first culture medium comprises from 2 pM to about 10 pM of CHIR-99021. In some embodiments, the second culture medium comprises 50 ng / ml VEGF and 10 ng / ml bFGF. In other embodiments, the second culture medium further comprises Y-27632 and SB-431542. In various embodiments, the third culture medium comprises 10 pM Y-27632 and 1 pM SB-431542. In certain embodiments, the third culture medium further comprises VEGF and bFGF. In particular instances, the first culture medium and / or the second medium is absent of insulin.

[0278] Some aspects of the invention utilize a dopaminergic neuron (DN) in combination with a CD200R engagement strategy. In other aspects, the DN cell is derived from a HIP cell, a HIPO- cell, or overexpresses CD 16, CD32, or CD64.RUC027WSF2024-223

[0279] In some aspects, the isolated dopaminergic neuron is selected from the group consisting of a neuronal stem cell, neuronal progenitor cell, immature dopaminergic neuron, and mature dopaminergic neuron.

[0280] In some aspects, provided herein is a method of treating a patient suffering from a neurodegenerative disease or condition. In some embodiments, the method comprises administering a composition comprising a therapeutically effective amount of a population of DN cells with CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecules. In some aspects, the composition further comprises a therapeutically effective carrier. In some aspects, the population of the isolated hypoimmune dopaminergic neurons is on a biodegradable scaffold. The administration may comprise transplantation or injection. In some aspects, the neurodegenerative disease or condition is selected from the group consisting of Parkinson’s disease, Huntington disease, and multiple sclerosis.

[0281] In some aspects, the invention provides a method of producing a population of the DN cells by in vitro differentiation. In some embodiments, the endogenous P-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, CD47 expression has been increased, the blood group is O and Rh-, or CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecules are overexpressed. In some aspects, the method comprises (a) culturing the population of cells in a first culture medium comprising one or more factors selected from the group consisting of sonic hedgehog (SHH), BDNF, EGF, bFGF, FGF8, WNT1, retinoic acid, a GSK3P inhibitor, an ALK inhibitor, and a ROCK inhibitor to produce a population of immature dopaminergic neurons; and (b) culturing the population of immature dopaminergic neurons in a second culture medium that is different than the first culture medium to produce a population of dopaminergic neurons.

[0282] In some embodiments, the GSKP inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSKP inhibitor is at a concentration ranging from about 2 pM to about 10 pM. In some embodiments, the ALK inhibitor is SB- 431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 1 pM to about 10 pM. In some embodiments, the first culture medium and / or second culture medium are absent of animal serum.

[0283] In some aspects of the invention, the method also comprises isolating the population of DN cells from non-DN cells. In some apsects, the method further comprises cry opreserving the isolated population of hypoimmune dopaminergic neurons.RUC027WSF2024-223

[0284] Some aspects of the invention utilize an isolated engineered hypoimmune pancreatic islet cell in combination with CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecules. In other aspects, the pancreatic islet cell is derived from a HIP cell, a HIPO- cell, or overexpresses CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecules.

[0285] In some embodiments, the isolated hypoimmune pancreatic islet cell is selected from the group consisting of a pancreatic islet progenitor cell, immature pancreatic islet cell, and mature pancreatic islet cell.

[0286] In some aspects, provided herein is a method of treating a patient suffering from diabetes. The method comprises administering a composition comprising a therapeutically effective amount of a population of any one of the pancreatic islet cells described herein in combination with a CD200R engagement, CD99 ligand engagement, and / or Fc sequestration strategy. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of the isolated hypoimmune pancreatic islet cells is on a biodegradable scaffold. In some instances, the administration comprises transplantation or injection.

[0287] In some aspects, provided herein is a method of producing a population of hypoimmune pancreatic islet cells from a population of CD200R engager, CD99 ligand engager, and / or Fc sequestration molecule overexpressing cells by in vitro differentiation. In some embodiments, the endogenous P-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated, CD47 expression has been increased, the blood type is O and Rh- in the HIPO- cells. The method comprises: (a) culturing the population of CD200R engager, CD99 ligand engager, and / or Fc sequestration molecule-overexpressing cells in a first culture medium comprising one or more factors selected from the group consisting insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (EGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), sonic hedgehog (SHH), and vascular endothelial growth factor (VEGF), transforming growth factor-P (TGFP) superfamily, bone morphogenic protein-2 (BMP2), bone morphogenic protein-7 (BMP7), a GSK3P inhibitor, an ALK inhibitor, a 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 culture medium that is different than the first culture medium to produce a population of hypoimmune pancreatic islet cells.RUC027WSF2024-223

[0288] In some embodiments, the GSK inhibitor is CHIR-99021, a derivative thereof, or a variant thereof. In some instances, the GSK inhibitor is at a concentration ranging from about 2 pM to about 10 pM. In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 1 pM to about 10 pM. In some embodiments, the first culture medium and / or second culture medium are absent of animal serum.

[0289] In some embodiments, the method also comprises isolating the pancreatic islet cells from non-pancreatic islet cells. In some embodiments, the method further comprises cry opreserving the isolated population of hypoimmune pancreatic islet cells.

[0290] In some aspects, provided herein is an isolated, engineered hypoimmune retinal pigmented epithelium (RPE) cell in combination with a CD200R engagement strategy. In other aspects, the RPE cell differentiated from a HIP cell, a HIPO- cell, a CIP cell, or overexpresses CD 16, CD32, or CD64.

[0291] In some embodiments, the isolated hypoimmune RPE cell is selected from the group consisting of a RPE progenitor cell, immature RPE cell, mature RPE cell, and functional RPE cell.

[0292] In some aspects, provided herein is a method of treating a patient suffering from an ocular condition. The method comprises administering a composition comprising a therapeutically effective amount of a population of any one of a population of the isolated RPE cells described herein in combination with a CD200R engagement strategy. In some embodiments, the composition further comprises a therapeutically effective carrier. In some embodiments, the population of the isolated hypoimmune RPE cells is on a biodegradable scaffold. In some embodiments, the administration comprises transplantation or injection to the patient’s retina. In some embodiments, the ocular condition is selected from the group consisting of wet macular degeneration, dry macular degeneration, juvenile macular degeneration, Leber's Congenital Ameurosis, retinitis pigmentosa, and retinal detachment.

[0293] In some aspects, provided herein is a method of producing a population of RPE cells from a population of cells by in vitro differentiation. In some embodiments, the endogenous P-2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated and expression of a SIRPa engager like CD47 has been increased in the HIPO-cells. The method comprises: (a) culturing the population of HIPO-cells in a first culture medium comprising any one of the factors selected from theRUC027WSF2024-223 group consisting of activin A, bFGF, BMP4 / 7, DKK1, IGF1, noggin, a BMP inhibitor, an ALK inhibitor, a ROCK inhibitor, and a VEGFR inhibitor to produce a population of pre- RPE cells; and (b) culturing the population of pre-RPE cells in a second culture medium that is different than the first culture medium to produce a population of hypoimmune RPE cells.

[0294] In some embodiments, the ALK inhibitor is SB-431542, a derivative thereof, or a variant thereof. In some instances, the ALK inhibitor is at a concentration ranging from about 2 pM to about 10 pM. In some embodiments, the ROCK inhibitor is Y-27632, a derivative thereof, or a variant thereof. In some instances, the ROCK inhibitor is at a concentration ranging from about 1 pM to about 10 pM.

[0295] In some embodiments, the first culture medium and / or second culture medium are absent of animal serum.

[0296] In some embodiments, the method further comprises isolating the population of hypoimmune RPE cells from non-RPE cells. In some embodiments, the method further comprises cry opreserving the isolated population of hypoimmune RPE cells.

[0297] In one aspect, the therapeutic cells are comprehensively immune protected cells. In some embodiments, the human pluripotent stem cells (PSCs) are comprehensively immune protected cells. In some embodiments, they are hypoimmune pluripotent stem cells (hiPSC). They are rendered hypo-immunogenic by a) the disruption of the B2M gene at each allele (e.g. B2M - / -), b) the disruption of the CIITA gene at each allele (e.g. CIITA - / -), and c) by the overexpression of the CD47 gene (CD47+, e.g. through introducing one or more additional copies of the CD47 gene or activating the genomic gene). This renders the hiPSC population B2M- / - CIITA -I- CD47tg. In a preferred aspect, the cells are non-immunogenic. In another embodiment, the HIP cells are rendered non-immunogenic B2M- / - CIITA - / - CD47tg as described above but are further modified by including an inducible suicide gene that is induced to kill the cells in vivo when required. In other aspects, CD200R engager, CD99 ligand engager, and / or Fc sequestration molecule-overexpressing HIPO- cells are created when HIP cells are rendered blood type O by by knocking out the ABO gene Exon 7 or silencing the SLC14A1 (JK) gene and the cells are rendered Rh- by knocking out the C and E antigens of the Rh blood group system (RH), K in the Kell system (KEL), Fya and Fy3 in the Duffy system (FY), Jkb in the Kidd system (JK), or U and S in the MNS blood group system.RUC027WSF2024-223

[0298] Alpha-1 antitrypsin deficiency (A1AD) is a genetic disorder that may result in lung disease or liver disease and is caused by a mutation in the SERPINA1 gene. The deficiency in Al AT leads to an imbalance with high neutrophil elastase activity, an enzyme that can disrupt connective tissue in the lung. Individuals with A1AD may develop chronic obstructive pulmonary disease or emphysema. Treatment of advanced disease includes intravenous infusions of the Al AT protein derived from donated human plasma or lung transplantation. The invention provides a cell therapy product to replace the missing Al AT and prevent progression of the disease. In some embodiments, the Al AT therapy maintains physiologic Al AT levels and prevents morphologic and functional diseases, e.g. in the lungs.

[0299] Thus, the invention provides an isolated, engineered hypoimmune cell that expresses a therapeutic protein. In some aspects of the invention, that therapeutic protein is Al AT. In other aspects, the therapeutic protein, e.g. Al AT, is transplanted into a subject in combination with a CD200R engagement strategy. In other aspects, the hypoimmune cell expressing the therapeutic protein is, or is differentiated from, a HIP cell, a HIPO- cell, or overexpresses CD 16, CD32, or CD64.F. Maintenance of Pluripotent Cells

[0300] Once generated, the pluripotent cells, such as HIP, HIPO-, CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecule cells, can be maintained in an undifferentiated state as is known for maintaining iPSCs. For example, HIP or CIP cells are cultured on Matrigel using culture media that prevents differentiation and maintains pluripotency.G. Differentiation of Pluripotent Cells

[0301] The invention provides pluripotent cells, such as HIP, HIPO-, CIP, CD200R engagers, CD99 ligand engagers, and / or Fc sequestration molecule cells that are differentiated into different cell types for subsequent transplantation into subjects in combination with a CD200R engageement, CD99 ligand engagement, and / or Fc sequestration strategy. As will be appreciated by those of skill in the art, the methods for differentiation depend on the desired cell type using known techniques. The cells are differentiated in suspension and then put into a gel matrix form, such as matrigel, gelatin, or fibrin / thrombin forms to facilitate cell survival. Differentiation is assayed as is known in the art, generally by evaluating the presence of cell-specific markers.

[0302] In some embodiments, the pluripotent cells are differentiated into hepatocytes to address loss of the hepatocyte functioning or cirrhosis of the liver. There are a number ofRUC027WSF2024-223 techniques that can be used to differentiate HIPO- cells into hepatocytes; see for example Pettinato et al., doi: 10.1038 / spre32888, Snykers et al., Methods Mol Biol 698:305-314 (2011), Si-Tayeb et al, Hepatology 51 :297-305 (2010) and Asgari et al., Stem Cell Rev (:493- 504 (2013), all of which are hereby expressly incorporated by reference in their entirety and specifically for the methodologies and reagents for differentiation. Differentiation is assayed as is known in the art, generally by evaluating the presence of hepatocyte associated and / or specific markers, including, but not limited to, albumin, alpha fetoprotein, and fibrinogen. Differentiation can also be measured functionally, such as the metabolization of ammonia, LDL storage and uptake, ICG uptake and release and glycogen storage.

[0303] In some embodiments, the pluripotent cells are differentiated into beta-like cells, islet cells, or organoids for transplantation to address type I diabetes mellitus (T1DM). Cell systems are a promising way to address T1DM, see, e.g., Ellis et al., doi / 10.1038 / nrgastro.2017.93, incorporated herein by reference. Additionally, Pagliuca et al. reports on the successful differentiation of P-cells from hiPSCs (see doi / 10.106 / j . cell.2014.09.040, hereby incorporated by reference in its entirety and in particular for the methods and reagents outlined there for the large-scale production of functional human P cells from human pluripotent stem cells). Furthermore, Vegas et al. shows the production of human P cells from human pluripotent stem cells followed by encapsulation to avoid immune rejection by the host; (doi: 10.1038 / nm.4030, hereby incorporated by reference in its entirety and in particular for the methods and reagents outlined there for the large-scale production of functional human P cells from human pluripotent stem cells).

[0304] Differentiation is assayed as is known in the art, generally by evaluating the presence of P cell associated or specific markers, including but not limited to, insulin. Differentiation can also be measured functionally, such as measuring glucose metabolism, see generally Muraro et al, doi: 10.1016 / j.cels.2016.09.002, hereby incorporated by reference in its entirety, and specifically for the biomarkers outlined there.

[0305] Once the differentiated beta cells are generated, they can be transplanted (either as a cell suspension or within a gel matrix as discussed herein) into the portal vein / liver, the omentum, the gastrointestinal mucosa, the bone marrow, a muscle, or subcutaneous pouches.

[0306] In some embodiments, the pluripotent cells are differentiated into retinal pigment epithelium (RPE) to address sight-threatening diseases of the eye. HumanRUC027WSF2024-223 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 in particular for the methods and reagents outlined there for the differentiation techniques and reagents; see also Mandai et al., doi: 10.1056 / NEJMoal608368, also incorporated in its entirety for techniques for generating sheets of RPE cells and transplantation into patients.

[0307] Differentiation can be assayed as is known in the art, generally by evaluating the presence of RPE associated and / or specific markers or by measuring functionally. See for example Kamao et al., doi: 10.1016 / j.stemcr.2013.12.007, hereby incorporated by reference in its entirety and specifically for the markers outlined in the first paragraph of the results section.

[0308] In some embodiments, the pluripotent cells are differentiated into cardiomyocytes to address cardiovascular diseases. Techniques are known in the art for the differentiation of hiPSCs to cardiomyoctes and discussed in the Examples. Differentiation can be assayed as is known in the art, generally by evaluating the presence of cardiomyocyte associated or specific markers or by measuring functionally; see for example Loh et al., doi: 10.1016 / j.cell.2016.06.001, hereby incorporated by reference in its entirety and specifically for the methods of differentiating stem cells including cardiomyocytes.

[0309] In some embodiments, the pluripotent cells are differentiated into endothelial colony forming cells (ECFCs) to form new blood vessels to address peripheral arterial disease. Techniques to differentiate endothelial cells are known. See, e.g., Prasain et al., doi: 10.1038 / nbt.3048, incorporated by reference in its entirety and specifically for the methods and reagents for the generation of endothelial cells from human pluripotent stem cells, and also for transplantation techniques. Differentiation can be assayed as is known in the art, generally by evaluating the presence of endothelial cell associated or specific markers or by measuring functionally.

[0310] In some embodiments, the pluripotent cells are differentiated into thyroid progenitor cells and thyroid follicular organoids that can secrete thyroid hormones to address autoimmune thyroiditis. Techniques to differentiate thyroid cells are known the art. See, e.g. Kurmann et al., doi : 10.106 / j . stem.2015.09.004, hereby expressly incorporated by reference in its entirety and specifically for the methods and reagents for the generation of thyroid cells from human pluripotent stem cells, and also for transplantation techniques. DifferentiationRUC027WSF2024-223 can be assayed as is known in the art, generally by evaluating the presence of thyroid cell associated or specific markers or by measuring functionally.H. Transplantation of CD200R Engager, CD99 Ligand Engager, and / or Fc Sequestration Molecule Positive Therapeutic Cells

[0311] As will be appreciated by those in the art, the differentiated pluripotent cell derivatives are transplated using techniques known in the art that depends on both the cell type and the ultimate use of these cells. In general, the cells of the invention are transplanted either intravenously or by injection at particular locations in the patient. When transplanted at particular locations, the cells may be suspended in a gel matrix to prevent dispersion while they take hold.

[0312] In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.VIII. EXAMPLES

[0313] HIP, HIPO-, CD16 / CD32 / CD64, and PMN-inhibitory cells are generated as disclosed in WO2018 / 132783, W02020 / 018620, W02020 / 018615, WO2020 / 231882, US11162079, WO2021 / 076427, and WO2021 / 146471. The foregoing are incorporated by reference herein in their entirety.Example 1: Immune Cell Inhibition by a CD200R Engager

[0314] CD200R is an inhibitory receptor on polymorphonuclear cells (PMNs), macrophages, and some additional immune cells, e.g., NK cells. The CD200R engager is a synthetic molecule expressed on engineered cells. The CD200R engager binds to CD200R and exerts agonistic function, which results in an inhibitory signal in that immune cell.(Figure 1)

[0315] A CD200R engager molecule was constructed having the sequence of SEQ ID NOV. It comprises an Anti-CD200R Variable Heavy Chain (VH) Domain (SEQ ID NO: 1) and an Anti-CD200R Variable Light Chain (VL) Domain (SEQ ID NOV). It was constructed in this example with a CD8a signal peptide (SEQ ID NOV) for expression purposes only. It further comprised the (GGGGS)s linker (SEQ ID NO:4), a CD8a hinge domain (SEQ ID NO:5), and a CD140a transmembrane domain (SEQ ID NO:6).RUC027WSF2024-223

[0316] Figure 2A shows flow cytometry analysis of CD200R Engager (CD200R-E) expression in transduced wild-type human iPSC-derived endothelial cells (iECs). Figure 2B shows the CD200R Engager expression in DKO SIRPa-E iECs. These cells are HLA class I and Il-deficient (double knockout; DKO) and express the SIRPa Engager. The lentiviral particles for transduction of the CD200R Engager transgene also included a Red Fluorescent Protein (RFP) tag. The flow cytometry histogram in both panels show RFP expression in untransduced WT iECs or DKO SIRPa-E iECs (left peak) and transduced WT iECs +CD200R-E or transduced DKO SIRPa-E iECs +CD200R-E (right peak). This confirmed their expression of the CD200R Engager.

[0317] Isolated human PMNs. Human PMNs were isolated from fresh blood. In a 15 ml conical tube, Leuko-Spin and Lympho-Spin (PluriSelect, Leipzig, Germany, Cat. Nos. SKU 60-00091-10 and SKU 60-00092-10) were mixed in a ratio of 1 : 1. Fresh blood was layered on top of the Leuko-Lympho-Spin mix followed by centrifugation for 30 min at 1000g with the brake off. After the density centrifugation, PMNs were extracted from the lower layer.

[0318] DAMPs activate PMNs. PMNs were activated via damage-associated molecular patterns (DAMPs) that are endogenous pro-inflammatory proteins, nucleic acids, or lipids commonly released by dying graft cells after surgical trauma, ischemia, and inflammation. Cellular stress and tissue injury induce perturbations of the microenvironment and activate the innate immune system. DAMPs recruit PMNs from the blood to sites of tissue damage where PMNs contribute to wound healing. PMNs subsequently release proinflammatory cytokines and orchestrate a diverse immune response. PMNs were activated by DAMPs in cytotoxicity assays in vitro using sonicated cell debris that was added to the target cells together with the PMNs.

[0319] BLI killing assay over 2 hours with freshly isolated human PMNs. Human Wildtype iECs (WT iECs) express Firefly luciferase for the BLI killing assays. Human WT iECs (Figure 3A) or human WT iECs expressing the CD200R Engager (Figure 3B) were plated and let attach for 16 hours. Then, human PMNs or PMNs together with sonicated cell debris (DAMPs) of the same target cells were added to the wells and let incubate for 2 hours. The percent BLI signal after the 2 hours incubations is shown. PMNs alone did not cause any target cell killing. When DAMPs were added, however, WT iECs were partially killed by PMNs. This PMN killing was mitigated when the target cells expressed the CD200R Engager.RUC027WSF2024-223

[0320] BLI killing assay over 2 hours with freshly isolated human polymorphonuclear cells (PMNs). Firefly luciferase-expressing human DKO SIRPa-E iECs (Figure 4A) or human DKO SIRPa-E iECs expressing the CD200R Engager (Figure 4B) were plated and let attach for 16 hours. Then, human PMNs or PMNs together with sonicated cell debris (DAMPs) of the same target cells were added to the wells and let incubate for 2 hours. The percent BLI signal after the 2 hours incubation is shown. PMNs alone did not cause any target cell killing. When DAMPs were added, however, CIP iECs were partially killed by PMNs. This PMN killing was mitigated when the target cells expressed the CD200R Engager.

[0321] BLI killing assay over 6 hours with human peripheral blood NK cells or human macrophages (MAC). Firefly luciferase-expressing human DKO iECs (Figure 5A) or human DKO iECs expressing the CD200R Engager (Figure 5B) were plated and let attach for 16 hours. Then, human NK cells or MACs were added to the wells and let incubate for 6 hours. The percent BLI signal after the 6 hours incubation is shown. DKO iECs were killed by NK cells or MACs. DKO iECs expressing the CD200R Engager were largely protected from NK cell killing and MAC killing.Example 2: Overexpressing CD200 and CD99 Prevented Rhesus monkey killing of human iECs

[0322] Human cells were killed by PMNs when transplanted into into Neu5Gc- expressing species such as rhesus monkeys, cynomolgus monkeys, or C57BL / 6 mice. Here, CD200 and CD99 overexpression protected cells from PMN killing in the presence of a Neu5Gc / Neu5Ac mismatch.

[0323] Induced wt or HIP ECs from human (Figure 6A), and C57BL / 6 mouse, or rhesus monkey (Figure 6B) were plated on the XCelligence platform and were incubated with PMNs from a species with a Neu5 Ac / Neu5Gc mismatch. The PMNs were not stimulated. In all combinations, the target cells were expeditiously killed by unstimulated xenogeneic PMNs.

[0324] Figure 7 shows that the above phenomenon is xeno-specific. Killing of allogeneic ECs by unstimulated PMNs is usually not observed. Rhesus monkey ECs were not killed by unstimulated allogeneic rhesus monkey PMNs. DAMPs or macaque IL-2 stimulated allogeneic PMNs, however, did induce killing.RUC027WSF2024-223

[0325] In order to mitigate human iEC susceptibility to rhesus monkey PMN killing, the missing CMAH gene was introduced into cells that lack its expression. Human rhHIP iECs (human cells with B2M- / - and CIITA- / - and expressing rhesus monkey CD47) were transduced to express CMAH using lentiviral particles. These human rhHIP CMAH tg iECs were then challenged with rhesus monkey PMNs on the XCelligence platform. Figure 8 shows that CMAH replacement alone did not alleviate xenogeneic PMN killing.

[0326] Figure 9 shows that CMAH expression combined with CD200 and CD99 overexpression in human rhHIP cells mitigated rhesus monkey PMN killing. CMAH transduction was combined with overexpression of CD200 and CD99 using lentiviral particles. When these engineered human rhHIP CD200 CD99 CMAH tg iECs were challenged with rhesus monkey PMNs, target cell killing was prevented. This was observed even when the PMNs were stimulated with sonicated cells or macaque IL-2.Example 3: Engineered pig cells for xenotransplantation into humans without immunosuppression

[0327] Currently, xenogeneic cell or organ transplantation into humans requires immunosuppression co-therapy to prevent rejection. The invention provides for the first time an adapted comprehensive hypoimmune strategy for xenotransplation. It is exemplified here for transplanting pig cells into humans. As shown in Figure 10, the engineered pig cells in this example utilize depleted SLA-I and SLA-II function or expression, a CD47 chimera that does not carry any CD47 signalling motifs for intracellular signalling, as well as truncated CD64, CD99, and CD200.

[0328] In particular, this example provides adapted swine comprehensively immune protected cells for human transplantation. Both the B2m and Ciita genes were knocked out to deplete swine leukocyte antigen (SLA) class I and II to prevent T cell activation. A chimera containing a truncated human CD47 extracellular domain fused to the CD 140a transmembrane domain (CD47.CD140a) was expressed to inhibit human NK cells and macrophages. A chimera containing a truncated human CD64 extracellular domain fused to the CD140a transmembrane domain (CD64.140a) was expressed to protect the cells from human IgG antibody attack. The human CD99 and CD200 proteins were expressed that interact with inhibitory receptors on human PMNs to prevent PMN-mediated killing of the pig xenograft cells.RUC027WSF2024-223

[0329] Figure 11A shows Pig wt endothelial cells (ECs) that were incubated with human PMNs. They were either unstimulated or stimulated with cell debris (DAMPs) or human IL-2. The pig target cells were transduced to express firefly luciferase and their bioluminescence signal (BLI signal) was recorded over time. The relative BLI signal after 24 hours is plotted in the graph. Human PMNs, whether unstimulated or stimulated, killed the pig wt target cells.

[0330] Figure 11B shows pig wt ECs that were incubated with primed human PBMCs. The human PBMCs were primed by incubating them with pig wt ECs for 10 days in vitro. The human PBMCs were then sorted for CD45 expression, thus excluding the pig ECs, resulting in an enriched primed human PBMC population. These primed PBMCs were then added to the pig wt ECs. After 24 h, the pig wt EC target cells had lost their BLI signal, indicating that they were killed.

[0331] Figure 11C shows 50,000 firefly luciferase-positive pig wt ECs injected into xenogeneic humanized mice together with 1 million human PMNs and 1 mg anti -MICA IgGl antibody. On days 1 and 2, another dose of 1 mg anti -MIC A IgGl antibody was injected into the EC grafts. Cell survival was assessed by serial bioluminescence imaging. As shown, all four pig wt EC grafts were quickly killed and their BLI signals vanished in just 2 days.

[0332] Pig wt ECs were engineered to express human CD99 and CD200 (wt.99.200 ECs). Figure 12A shows that when these cells were incubated with unstimulated and stimulated human PMNs, no target cell killing was observed. When they were incubated with primed human PBMCs, the pig target cells were killed (Figure 12B).

[0333] Pig ECs were engineered to deplete SLA class I and II through the knockout of the B2m and Ciita genes. Additionally, a truncated CD47.140a chimera, CD99, and CD200 were expressed. Figure 13A shows that when incubated with unstimulated and stimulated human PMNs, no target cell killing was observed. Figure 13B shows that when the pig B2m' / 'Ciita' / '.tCD47.99.200 ECs were incubated with primed human PBMCs, the pig target cells survived and the BLI signal stayed stable.

[0334] Figure 13C shows that when the pig B2m' / 'Ciita' / '.tCD47.99.200 ECs were incubated with a cytotoxic anti-MICA IgGl antibody and human NK cells, the target cells were killed.

[0335] Pig B2m' / 'Ciita' / '.tCD47.tCD64.99.200 ECs were engineered to also express the truncated CD64.140a Fc receptor. Figure 14A shows that when these cells are incubatedRUC027WSF2024-223 with unstimulated and stimulated human PMNs, no target cell killing was observed. Figure 14B shows that when pig B2m' / 'Ciita' / '.tCD47.tCD64.99.200 ECs were incubated with primed human PBMCs, no target cell killing was observed. Figure 14C shows that when the pig B2nr / 'Ciita' / '.tCD47.tCD64.99.200 ECs were incubated with a cytotoxic anti-MICA IgGl antibody and human NK cells, the BLI signal remained stable and no target cell killing was observed.

[0336] Figure 14D shows 50,000 firefly luciferase-positive pig B2m' / 'Ciita' / ‘ .tCD47.tCD64.99.200 ECs injected into xenogeneic humanized mice together with 1 million human PMNs and 1 mg anti-MICA IgGl antibody. On days 1 and 2, another dose of 1 mg anti-MICA IgGl antibody was injected into the EC grafts. Cell survival was assessed by serial bioluminescence imaging. As shown, all four pig B2m' / 'Ciita' / '.tCD47.tCD64.99.200 EC grafts for at least about 40 days and their BLI signals remained stable and even increased over time suggesting continued proliferation of the ECs.METHODS

[0337] Primary cells. Rhesus primary ECs were purchased from ATCC (CRL-1780, cat.no. RF / 6A, ATCC, Manassas, VA) and cultured in Eagle's Minimum Essential Medium (ATCC) containing 10% FCS hi and 1% pen / strep (both Gibco). Media was changed twice per week. Pig primary ECs were purchased from Cell Biologies (cat.no. P-6065, Cell Biologies, Chicago, IL) and cultured in complete endothelial cell medium (Cell Biologies) on gelatin coated T75 flasks. Media was changed every 2 days and TrypLE (ThermoFisher, Waltham, MA) was used for cell passaging with a ratio of 1 :4 every 5 days. Rhesus and cynomolgus PMNs were provided by the Alpha Genesis Primate Research Center.

[0338] Derivation of mouse iPSCs. Mouse tail tip fibroblasts were dissociated and isolated with collagenase type IV (Life Technologies) and maintained with Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 1% glutamine, 4.5 g / 1 glucose, 100 U / ml penicillin, and 100 pg / ml streptomycin (pen-strep) at 37 °C, 20% O2, and 5% CO2 in a humidified incubator. One million mouse fibroblasts were then reprogrammed using a mini-intronic plasmid carrying sequences of Oct4, Klf4, Sox2 and c- Myc as well as short hairpin RNA against p53 (10-12 pM of DNA) using the Neon Transfection system. After transfection, fibroblasts were plated on mitomycin-inhibited CF1 mouse embryonic fibroblasts (MEF, Applied Stemcell) and kept in fibroblast media with the addition of sodium butyrate (0.2 mM) and 50 pg / ml ascorbic acid. When ESC-like colonies appeared, media was changed to mouse iPSC media containing DMEM + GlutaMax 31966RUC027WSF2024-223(Gibco) with 10% heat-inactivated fetal calf sera (FCShi), 1% MEM-NEAA and 1% pen- strep (all Gibco). With every passage, the iPSCs were sorted for the mouse pluripotency marker SSEA-1 using antibody-coated magnetic bead-based cell sorting.

[0339] Generation of B2m- / “ mouse iPSCs. CRIPSR technology was used for disruption of the B2m gene. For targeting the coding sequence of the mouse B2m gene, the CRISPR sequence 5'-TTCGGCTTCCCATTCTCCGG(TGG)-3' was annealed and ligated into the All-In-One (AIO) vectors containing the Cas9 expression cassette as per the kit’s instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fisher). Mouse iPSCs were transfected with the AIO vectors using Neon electroporation with two 1,200 V pulses of 20 ms duration. The transfected iPSC cultures were dissociated to single cells using 0.05% trypsin (Gibco) and then sorted with FACSAria cell sorter (BD Bioscience) for removing doublets and debris by selective gating on forward and side light scatter properties. Single cells were expanded to full-size colonies and tested for CRISPR editing by screening for the presence of the altered sequence from the CRISPR cleavage site. Briefly, the target sequence was amplified via PCR using AmpliTaq Gold Master Mix (Applied Biosystems) and the primers B2m gDNA forward: 5'-CTGGATCAGACATATGTGTTGGGA-3', reverse: 5'- GCAAAGCAGTTTTAAGTCCACACAG-3'. After cleanup of the obtained PCR product (PureLink Pro 96 PCR Purification Kit, Thermo Fisher), Sanger sequencing was performed. The Ion Personal Genome Machine (PGM) Sequencing was used for the identification of the homogeneity, through sequencing of a PCR amplified 250 base pair region of the B2m gene using primers B2m gDNA PGM forward: 5'-TTTTCAAAATGTGGGTAGACTTTGG-3' and reverse: 5'-GGATTTCAATGTGAGGCGGGT-3'. The PCR product was purified as described above and prepared using the Ion PGM Hi-Q Template Kit (Thermo Fisher). Analyses were performed on the Ion PGM System with the Ion 318 Chip Kit v.2 (Thermo Fisher).

[0340] Generation of B2m- / _Ciita_ / “ mouse iPSCs. CRIPSR technology was used for the further disruption of the Ciita gene. For targeting the coding sequence of mouse Ciita gene, the CRISPR sequence 5’ - GGTCCATCTGGTCATAGAGG (CGG)-3' was annealed and ligated into the All-In-One (AIO) vectors containing the Cas9 expression cassette as per the kit’s instructions (GeneArt CRISPR Nuclease Vector Kit, Thermo Fisher). miPSC were transfected with the AIO vectors using the same condition for B2m disruption. The transfected miPSC cultures were dissociated to single cells using 0.05% trypsin (Gibco) and then sorted with FACSAria cell sorter (BD Bioscience) for removing doublets and debris byRUC027WSF2024-223 selective gating on forward and side light scatter properties. Single cells were expanded to full-size colonies and tested for CRISPR editing by screening for the presence of an altered sequence from the CRISPR cleavage site. Briefly, the target sequence was amplified via PCR using AmpliTaq Gold Master Mix (Applied Biosystems) and the primers Ciita gDNA forward: 5'-CCCCCAGAACGATGAGCTT-3', reverse: 5'- TGCAGAAGTCCTGAGAAGGCC-3'. After cleanup of the obtained PCR product (PureLink Pro 96 PCR Purification Kit, Thermo Fisher), Sanger sequencing was performed. Using the DNA sequence chromatogram, edited clones were then identified through the presence of altered sequence from the CRISPR cleavage site. Indel size was calculated using the TIDE tool. PCR and ICC were performed again to verify the pluripotency status of the cells.

[0341] Generation of B2m_ / _Ciita“ / _Cd47 tg mouse iPSCs. Cd47 transgene overexpression was generated using lentivirus-mediated delivery of a Cd47-expressing vector containing the antibiotic resistance cassette blasticidin. The Cd47 cDNA was synthesized and cloned into the lentiviral plasmid pLenti6 / V5 (Thermo Fisher) with a blasticidin resistance. Sanger sequencing was performed to verify that no mutation had occurred. Lentivirus generation was performed with a stock titer of ten million TU per ml. The transduction wasperformed into 200,000 B2m Ciita mouse iPSCs, grown on blasticidin-resistant MEF cells for 72 h with a MOI ratio of 1 : 10 followed by antibiotic selection with 12.5 pg / ml blasticidin for 7 days. Antibiotic-selected pools were tested by RT-quantitative PCR amplification of Cd47 mRNA and flow cytometry detection of Cd47 on the surface of the cells. After the confirmation of Cd47, cells were expanded and validated by running pluripotency assays.

[0342] Generation of B2m- / _Ciita- / _CD47 tg pig ECs. Pig HIP ECs were similarly engineered through B2m and Ciita knockout and human CD47 overexpression.

[0343] Transduction to express firefly luciferase. Target cells, including iPSCs or ECs, were transduced to express firefly luciferase (Flue). One hundred thousand mouse iPSCs were plated in one gelatin-coated six-well plates and incubated overnight at 37 °C at 5% CO2. The next day, media was changed and one vial of Flue lentiviral particles expressing luciferase II gene under reengineered EFla promotor (GenTarget) was added to 1.5 ml media. After 36 h, 1 ml of cell media was added. After further 24 h, complete media change was performed. After 2 days, luciferase expression was confirmed by adding D- luciferin (Promega). Signals were quantified with IVIS 200 (Perkin Elmer) in maximum photons / s / cm2 / sr.RUC027WSF2024-223

[0344] Derivation and characterization of mouse iPSC-derived endothelial cells (mouse iECs). Mouse iPSC were plated on gelatin in six-well plates and maintained in mouse iPSC media. After the cells reached 60% confluency, the differentiation was started and media was changed to RPMI-1640 containing 2% B-27 minus Insulin (both Gibco) and 5 pM CHIR-99021 (Selleckchem). On day 2, the media was changed to reduced media: RPMI- 1640 containing 2% B-27 minus Insulin (both Gibco) and 2 pM CHIR-99021 (Selleckchem). From day 4 to day 7, cells were exposed to RPMI-1640 EC media, RPMI-1640 containing 2% B-27 minus Insulin plus 50 ng / ml mouse vascular endothelial growth factor (mVEGF; R&D Systems), 10 ng / ml mouse fibroblast growth factor basic (mFGFb; R&D Systems), 10 pM Y-27632 (Sigma- Aldrich) and 1 pM SB 431542 (Sigma-Aldrich). Endothelial cell clusters were visible from day 7 and cells were maintained in EGM-2 SingleQuots media (Lonza) plus 10% FCS hi (Gibco), 25 ng / ml mVEGF, 2 ng / ml mFGFb, 10 pM Y-27632 (Sigma-Aldrich) and 1 pM SB 431542. The differentiation process was completed after 21 days and undifferentiated cells detached during the differentiation process. For purification, cells went through magnetic-activated cell separation (MACS) purification according to the manufacturer’s protocol using anti-CD15 m Ab-coated magnetic microbeads (Miltenyi) for negative selection. The highly purified mouse iECs in the flow-through were cultured in EGM-2 SingleQuots media plus supplements and 10% FCS hi. TrypLE was used for splitting the cells 1 :3 every 3-4 days. Their phenotype was confirmed by immunofluorescence for CD31 (ab28364, Abeam) and VE-Cadherin (sc-6458, Santa Cruz Biotechnology). Briefly, cells were fixed with 4% paraformaldehyde in PBS for 15 min. Cell membranes were permeabilized with Permeabilization solution (ASB-0102, Applied StemCell), followed by Blocking solution (ASB-0103, Applied StemCell) and incubation with the primary antibodies. For visualization, cells were incubated with secondary antibody conjugated with AF488 or AF555 (Invitrogen). After nuclei staining with DAPI, images were obtained and analyzed with a Leica SP5 laser confocal microscope (Leica).

[0345] Generation of human iPSCs. The Human Episomal iPSC Line was derived from CD34+ cord blood using a three-plasmid, seven-factor (SOKMNLT; SOX2, OCT4 (POU5F1), KLF4, MYC, NANOG, LIN28 and SV40L T antigen) EBNA-based episomal system by Thermo Scientific. This cell line has been shown to be free of all reprogramming genes. Gene editing of hiPSC. hiPSC underwent two gene-modification steps. In the first step, CRISPR technology was used for a combined targeting of the coding sequence of human B2M gene with the CRISPR sequence 5'-CGTGAGTAAACCTGAATCTT-3' and the coding sequence of human CIITA gene with the CRISPR sequence 5'-RUC027WSF2024-223GATATTGGCATAAGCCTCCC-3'. Linearized CRISPR sequence with T7 promoter were used to synthesize gRNA as per the kit’s instructions (MEGAshortscript T7 Transcription Kit, Thermo Fisher). The resulting in vitro transcription (IVT) gRNA was then purified via the MEGAclear Transcription Clean-Up Kit. For IVT gRNA delivery, cells were electroporated with 300 ng IVT gRNA using a Neon electroporation system at 1,200 V, 30 ms, 1 pulse into hiPSC stably expressing Cas9. After electroporation, edited hiPSC were expanded for single cell seeding: Human iPSC cultures were dissociated into single cells using TrypLE Express (Gibco) and stained with Alexa Fluor 488-conjugated anti-TRA-160 mAb and propidium iodide. A FACSAria II cell sorter (BD Biosciences) was used for the sorting and doublets and debris were excluded from seeding by selective gating on forward and side light scatter properties. Viable pluripotent cells were selected on the absence of propidium iodide and presence of Tral-60 staining. Single cells were then expanded into full- size colonies that were analyzed for CRISPR editing by sequencing. CRISPR-mediated cleavage was assessed using the GeneArt Genomic Cleavage Detection Kit (Thermo Fisher) for testing of the initial edited pools. For screening of the isolated clones, genomic DNA was isolated from one million human iPSCs. The B2M and CIITA genomic DNA regions were PCR amplified using AmpliTaq Gold 360 Master Mix and the primer sets forward: 5'- TGGGGCCAAATCATGTAGACTC-3' and reverse: 5'- TCAGTGGGGGTGAATTCAGTGT-3' for B2M as well as forward: 5'- CTTAACAGCGATGCTGACCCC-3' and reverse: 5'-TGGCCTCCATCTCCCCTCTCTT-3' for CIITA. For TIDE analysis, the obtained PCR product was cleaned up (PureLink PCR Purification Kit, Thermo Fisher) and Sanger sequencing was performed for the prediction of indel frequency. After the confirmation of B2M and CIITA disruption, cells were further characterized through karyotype analysis and the TaqMan human iPSC Scorecard Panel (Thermo Fisher). The human iPSCs were found to be pluripotent and maintained a normal (46, XX) karyotype during the genome editing process. In the second step, the CD47 cDNA was synthesized and the DNA was cloned into a lentiviral plasmid with an EFS promotor and puromycin resistance cassette. Cells were transduced with lentiviral stocks and 8 pg / ml of Polybrene (Thermo Fisher). Media was changed daily after transduction. Three days after transduction, cells were expanded and selected with 0.5 pg / ml of puromycin. After 5 days of antibiotic selection, antibiotic-resistant colonies emerged and were further expanded to generate stable pools. The expression of CD47 transcripts was confirmed by quantitative PCR. Pluripotency assay (TaqMan hPSC Scorecard Panel, Thermo Fisher) and karyotyping was performed again to verify the pluripotent status of the cells.RUC027WSF2024-223

[0346] Human iPSC differentiation into human iECs. Human iPSC were plated on diluted Matrigel (356231, Corning) in six-well plates and maintained in Essential 8 Flex media (Thermo Fisher). The differentiation was started at 60% confluency and media was changed to RPMI-1640 containing 2% B-27 minus insulin (both Gibco) and 5 pM CHIR- 99021 (Selleckchem). On day 2, the media was changed to reduced media: RPMI-1640 containing 2% B-27 minus insulin (Gibco) and 2 pM CHIR-99021 (Selleckchem). From day 4 to 7, cells were exposed to RPMI-1640 EC media, RPMI-1640 containing 2% B-27 minus insulin plus 50 ng / ml human vascular endothelial growth factor (VEGF; R&D Systems), 10 ng / ml human fibroblast growth factor basic (FGFb; R&D Systems), 10 pM Y-27632 (Sigma- Aldrich), and 1 pM SB 431542 (Sigma- Aldrich). Endothelial cell clusters were visible from day 7 and cells were maintained in EGM-2 SingleQuots media (Lonza) plus 10% FCShi (Gibco), 25 ng / ml VEGF, 2 ng / ml FGFb, 10 pM Y-27632 (Sigma-Aldrich) and 1 pM SB 431542 (Sigma- Aldrich). The differentiation process was completed after 14 days und undifferentiated cells detached during the differentiation process. For purification, cells were treated with 20 pM PluriSln-1 (StemCell Technologies) for 48 h. The highly purified ECs were cultured in EGM-2 SingleQuots media (Lonza) plus supplements and 10% FCS hi (Gibco). TrypLE Express was used for passaging the cells 1 :3 every 3-4 days.

[0347] Transduction of CD99, CD200, and CMAH. For CD99 and CD200 transduction, 100,000 pig wt ECs, pig B2m’ / _Ciita' / _.tCD47 ECs, or pig B2m’ / _Ciita'A,tCD47.tCD64 ECs were plated in one 6-well and incubated overnight at 37°C at 5% CO2. Alternatively, human B2M" ' CIITA" ' rhesus CD47 iECs were plated. The next day, media was changed and CD99 and CD200 lentiviral particles driven by the CMV promotor (Origene, Rockville, MD) were added to 2 ml media with a MOI of 20. Alternatively, CMAH lentiviral particles driven by the CMV promotor (Origene, Rockville, MD) were added to 2 ml media with a MOI of 20. Complete media change was performed after 48 h. Cells were flow-sorted for the CD99 and CD200 double-positive population (BD FACSAria, BD Biosciences, San Jose, CA).

[0348] Isolation of human, rhesus, cynomolgus, or mouse PMNs. Human, rhesus, cynomolgus, or mouse PMNs were isolated from fresh blood. In a 15 ml conical tube, Leuko- Spin and Lympho-Spin were mixed in a ratio of 1 :1 (both PluriSelect, Leipzig, Germany). Fresh blood was layered on top of the Leuko-Lympho-Spin mix, followed by centrifugation for 30 min at 1000g with brake off. After the density centrifugation, PMNs can be found on the lower layer.RUC027WSF2024-223

[0349] Ex vivo human T cell priming. Human PBMCs were obtained after Ficoll separation of human blood draws. For priming, one million human PBMCs were co-cultured with 5 x 105target iECs or ECs in gelatin-coated flasks. The media, which consisted of a 1 : 1 mixture of EC medium and PBMC medium, was changed every 3 days. After 10 days, the cells in suspension were harvested and sorted using an APC-conjugated mouse anti-human CD3 antibody (clone SP34-2, cat.no. 557597, BD Biosciences, concentration 0.01 mg / ml) together with the APC-conjugated IgGlK isotype-matched control antibody (clone MOPC-21, cat.no. 550854, BD Biosciences, concentration 0.01 mg / ml) and a BV421 -conjugated antihuman CD8 antibody (clone SKI, cat.no. 344748, BioLegend, concentration 0.005 mg / ml) together with the BV421 -conjugated IgGlK isotype-matched control antibody (clone MOPC- 21, cat.no. 400157, BioLegend, concentration 0.005 mg / ml). The CD3+CD8+cells were sorted using a FACSAria flow cytometer (BD Biosciences) and used for BLI killing assays.

[0350] In vitro BLI killing assay. Fluc+ target iECs or ECs were counted and plated at a concentration of one thousand cells per 96-well plate. Then, target cells were mixed with PMNs or PBMCs at an E:T ratio of 10: 1. For ADCC assays, 1 pg / ml anti-MICA IgGl antibody (Creative Biolabs, TAB-0799CL) and human NK cells at an E:T ratio of 10: 1 were added. After 24 h in the BLI killing assay, luciferase expression was detected by adding D- luciferin (Promega, cat.no. P 1041 ). As controls, target cells were left untreated in cellspecific media. Signals were quantified with Ami HT (Spectral Instruments Imaging) in p / s / cm2 / sr and the relative BLI signal at 24 h was calculated.

[0351] Cytotoxicity assays on the XCelligence platform. In vitro killing assays were performed on the XCelligence SP platform and MP platform (ACEA BioSciences, San Diego, CA). Special 96-well E-plates (ACEA BioSciences) were coated with collagen (Sigma- Aldrich) and 40,000 target iECs or ECs were plated in 100 pl cell-specific medium. After the cell index (CI) reached 0.7, PMNs were added with an effector-to-target cell ratio of 1 : 1. PMNs, if indicated, were stimulated with 1 pg / ml human IL-2 or sonicated target cells (ratio 1 : 1 with the plated target cells). Data were standardized and analyzed with the RTCA software (ACEA BioSciences).Example 4: Comprehensively Immune Protected (CIP) pancreatic islet cells for diabetes treatment

[0352] Gene edited human primary pancreatic islets. All islets underwent the same procedure, but WT cells were not incubated with Cas9, guides, or lentiviral vectors. CIP isletRUC027WSF2024-223 cells underwent B2M knockout, CIITA knockout and were then incubated with a bicistronic lentivirus to deliver either SIRPaE.140a (an anti-SIRPa scFv / CD140a TMD fusion SEQ ID NO:27) and CD64.140a (SEQ ID NO:24) or tCD47.140a (SEQ ID NO:23) and CD64.140a. CIP islets were sorted for HLA class I and II deficiency and transgene expression.

[0353] Wild-type, B2M- / - CIITA- / - SIRPaE.140a CD64.140a and B2M- / - CIITA- / - tCD47.140a CD64.140a (CIP) islet cell phenotypes were characterized by flow cytometry. HLA-I, HLA-II, CD47, SIRPaE, and CD64 was measured on the cell surface (Figure 15). The re-clustered islets (also called pseudo-islets) released insulin in vitro in 5.8 mM glucose medium. The engineering did not impede the endocrine competence of the islets to sense glucose and release insulin. (Figure 16).

[0354] The CIP pancreatic islet cells survive allogeneic transplantation. Wildtype (WT) islet cells, B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+, or B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ islet cells were injected into 3 allogeneic humanized mice each. After 6 days, the spleens were recovered and splenocytes were isolated for ELISpot assays and serum was recovered for the detection of antibodies. The wild-type group evoked strong T cell activation as usually seen in fully allogeneic transplants into humanized mice. In stark contrast, both engineered hypoimmune islets did not show any T cell activation above background (Figure 17). Serum from the injected mice were incubated with the same islet cell fraction that was injected. A secondary anti-human IgM antibody was used to detect antibody bound to the islet cells. Donor islet-specific antibodies bound to the wild-type islet cells. No antibody-binding was observed, however, for both CIP islet groups. (Figure 18).

[0355] The CIP pancreatic islet cells resisted innate immune killing. Wild-type (WT) and DKO (B2M- / - CIITA- / -) islet cells that were HLA class I and II deficient but did not express any transgenes as well as both CIP islets B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+, and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ were incubated with primary human NK cells or macrophages. The wild-type cells remained unaffected by NK cells and macrophages. The DKO islets were killed expeditiously. Both hypoimmune CIP islet cells, however, resisted innate immune cells and showed no killing at all (Figure 19).

[0356] The CIP islets treated diabetes in diabetic humanized mice. Humanized mice received intraperitoneal Streptozotocin (STZ) injections (60 mg / kg; Sigma- Aldrich Cat. No. 572201) 6 days before injection of 450 islets. Blood was regularly drawn after transplantation and blood glucose was measured. In addition, all islet grafts were fireflyRUC027WSF2024-223 luciferase positive and graft survival was followed using BLI imaging. Wild-type islet cell grafts were rejected within 10 days in allogeneic, diabetic humanized mice. Blood glucose levels, which were >400 mg / dl at the time of transplantation, did not improve and remained steadily high. In contrast, both CIP islet grafts survived in their allogeneic hosts as indicated by the stable BLI signals during follow up. Also, both CIP islets were able to lower blood glucose levels over time, almost reaching levels of healthy animals. (Figure 20).

[0357] The CIP islet cells resisted ADCC and CDC. In addition to the two CIP islet cells tested above, which have two transgenes each, islets that are HLA class I and II depleted and express only either SIRPaE.140a or tCD47.140a but not CD64.140a were used. This showed the contribution of the CD64.140a transgene for the protection against antibody- mediated killing. Since EGFR is constitutively expressed on all islet cells, an FDA-approved anti-EGFR IgGl antibody (cetuximab) was used to induce in vitro killing by either NK cells (antibody-mediated cellular cytotoxicity, ADCC) or complement (complement-dependent cytotoxicity, CDC). All islet cells showed stable growth in vitro without intervention. When cetuximab with NK cells or cetuximab with serum was added, the wild-type and both engineered islet cells (not expressing the CD64.140a transgene) were quickly killed. Only the fully engineered B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+, and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ CIP islets were fully protected against antibody-mediated killing (Figure 21).

[0358] Antibody-mediated killing was assessed next in vivo. The same groups were injected into diabetic NSG mice. With the injection, they received 1 mg of cetuximab and one million human NK cells. On days 1 and 2, another 1 mg cetuximab was injected into the initial islet graft sites to ensure a very high antibody concentration in the immediate vicinity of the grafts. All grafts were positive for firefly luciferase and graft survival was monitored by BLI imaging. Blood glucose was followed to assess the ability of the islet grafts to treat diabetes. The WT islet grafts and both engineered islet grafts without CD64.140a were rejected over a few days and had no impact on blood glucose levels. Both fully engineered B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+, and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ CIP islet cells, however, were protected against antibody-mediated killing, showed stable BLI signals during follow-up, and had normalized blood glucose levels at the end of the study period (Figure 22).RUC027WSF2024-223Example 5: CIP islet cells survive humoral immunity

[0359] Patients with type 1 diabetes mellitus can have antibodies against beta cells including those directed against Zinc Transporter 8 (Znt8). CIP Beta cells showed reduced CDC killing susceptibiltiy in human sera from patients with type 1 diabetes mellitus.

[0360] In vitro human WT islet cells, B2M- / - CIITA- / - tCD47.140a+ islet cells, and and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ CIP islet cells were incubated in patient sera. Figure 23A shows CDC killing assays with sera from 2 patients with type 1 diabetes mellitus that did not have detectable Znt8 antibodies. All three islet cells, namely WT islet cells, B2M- / - CIITA- / - tCD47.140a+ islet cells, and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ islet cells, survived in this assay. In contrast, serum from a patient with type 1 diabetes mellitus having 11 pg / ml of Znt8 antibody (just above the 8 pg / ml test threshold) slowly killed WT islet cells and B2M- / - CIITA- / - tCD47.140a+ islet cells (Figure 23B). Only B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ CIP islet cells survived the CDC assay. Sera from two type 1 diabetes mellitus patients with high Znt8 antibody levels (98 pg / ml and 201 pg / ml) caused rapid CDC killing of WT islet cells and B2M- / - CIITA- / - tCD47.140a+ islet cells. Only B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ CIP CIP islet cells were fully protected. (Figure 23C.)

[0361] These assays showed that humoral immunity in type 1 diabetes mellitus patients is a real problem for cell replacement therapy. The hypoimmune CIP engineered cells of the invention that included an Fc sequestration receptor efficiently protected the cells from humoral immunity. The B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ islet cells were fully protected from Znt8 antibody killing even in sera with high antibody concentrations.Example 6: Hypoimmune edited stem cell-derived CIP islet cells treated diabetes

[0362] Gene edited human stem cell-derived islets. Human embryonic stem cells were differentiated into islet cells. All islet cells underwent the same procedure, but WT cells were not incubated with Cas9, guides, or lentiviral vectors. CIP islet cells underwent B2M knockout, CIITA knockout and were then incubated with a bicistronic lentivirus to deliver either SIRPaE.140a (SEQ ID NO:27) and CD64.140a (SEQ ID NO:24) or tCD47.140a (SEQ ID NO:23) and CD64.140a. CIP islets were sorted for HLA class I and II deficiency and transgene expression.RUC027WSF2024-223

[0363] Wild-type, B2M- / - CIITA- / - SIRPaE.140a CD64.140a and B2M- / - CIITA- / - tCD47.140a CD64.140a CIP islet cell phenotypes were characterized by flow cytometry. HLA-I, HLA-II, CD47, SIRPaE, and CD64 was measured on the cell surface (Figure 24). The clustered islets released insulin in vitro in 5.8 mM glucose medium. The engineering to CIP stem cell-derived islets (B2M- / - CIITA- / - SIRPaE.140a CD64.140a and B2M- / - CIITA- / - tCD47.140a CD64.140a) did not impede the endocrine competence of the islet cells to sense glucose and release insulin. (Figure 25).

[0364] The engineered stem cell-derived CIP islet cells treated diabetes in diabetic humanized mice. Humanized mice received intraperitoneal Streptozotocin (STZ) injections (60 mg / kg; Sigma-Aldrich Cat. No. 572201) 6 days before 1000 islets were injected. Blood was regularly drawn after transplantation and blood glucose was measured. In addition, all islet grafts were firefly luciferase positive and graft survival was followed using BLI imaging. Wild-type islet cell grafts were rejected within 10 days in allogeneic, diabetic humanized mice (Figure 26A). Blood glucose levels, which were >400 mg / dl at the time of transplantation, did not improve and remained steadily high over a period of 30 days. In contrast, both CIP islet grafts (B2M- / - CIITA- / - SIRPaE.140a CD64.140a and B2M- / - CIITA- / - tCD47.140a CD64.140a) survived in their allogeneic hosts over a period of at least about 30 days as indicated by the stable BLI signals during follow up. Also, both CIP islets were able to lower blood glucose levels over the 30-day measurement period. The blood glucose levels dropped to or below the 200 mg / dl level (Figure 26B and Figure 26C).

[0365] EGFR was constitutively expressed on WT and CIP stem cell-derived islet cells (Figure 27A). Cetuximab, an FDA-approved anti-EGFR IgGl antibody (IchorBio, Wayne, PA, cat.no. ICH4004) induces in vitro killing by either NK cells (antibody-mediated cellular cytotoxicity, ADCC) or complement (complement-dependent cytotoxicity, CDC). The engineered islet cells showed stable growth in vitro without intervention. When cetuximab with NK cells or cetuximab with serum was added, the WT islets were killed quickly. In contrast, the fully engineered B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+, and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ CIP islets were fully protected against antibody- mediated killing (Figure 27B).

[0366] Antibody-mediated killing was assessed next in vivo. WT islets, B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+, and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ islets were injected into diabetic NSG mice. With the injection, they received 1 mg of cetuximab and one million human NK cells. On days 1 and 2, another 1 mg cetuximab was injected intoRUC027WSF2024-223 the initial islet graft sites to ensure a very high antibody-concentration in the immediate vicinity of the grafts. All grafts were positive for firefly luciferase and graft survival was monitored by BLI imaging. Blood glucose was followed to assess the ability of the islet grafts to treat diabetes. The WT islet grafts were rejected over a few days and had no impact on blood glucose levels (Figure 28A). Both fully engineered B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+ and B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ CIP islet cells, however, were protected against antibody -mediated killing. They showed stable BLI signals for at least about 30 days or more post-engraftment. They also demonstrated normalizing blood glucose levels within about the first ten days post engraftment. They demonstrated normalized blood glucose levels by about 20 days and through at least about 30-days post-engraftment (Figures 28B and Figure 28C).

[0367] Methods

[0368] Gene editing of human primary islets. Human primary cadaveric islets were purchased from Prodo Labs and cultured overnight in PIM media (Prodo). The CRIPSR-Cas9 technology was used for the disruption of the B2M and CIITA genes. Islet clusters were dissociated in single cells using ACCUMAX (STEMCELL Technologies, Seattle, WA, Cat. No. 07921) for 10 min at 37 degrees C. The gRNA sequences 5'- CGTGAGTAAACCTGAATCTT-3' and 5'-GATATTGGCATAAGCCTCCC-3' were used for the human B2M gene and human CIITA gene, respectively. The Lonza P3 Primary Cell 4D-Nucleofector X Kit (V4XP-3032, Lonza) was used for the transfection of the islet cells. Briefly, cells were transduced with a final concentration of 50 million per ml in P3 buffer. Twenty microliters of the cell suspension were pipetted in one well of the 8-strip containing 13 pg of Cas9 enzyme and 6.5 pg of sgRNA, respectively. Lonza’s 4D-Nucleofector was used for the electroporation with the preset program CA-137. Islet cells were transferred into U-bottom 96-well plates containing 50,000 cells per well in PIM(S) media (Prodo) and rested for 1 hour at 37 degrees C and 5% CO2 before moving the plate on an orbital shaker for islet reclustering. A complete medium change was performed after 48 hours, and islet clusters were incubated on the orbital shaker for another 24 hours. Islet clusters were dissociated again into single cells using ACCUMAX for cell sorting using the anti-HLA-A,B,C antibody (clone G46 2.6, BD Biosciences, Catalog No. 555553) or IgGl isotype-matched control antibody (clone MOPC-21, BD Biosciences, Catalog No. 556650) and anti-HLA-DR,DP,DQ antibody (clone Tu39, BD Biosciences, Catalog No. 550853) or IgG2a isotype-matched control antibody (clone G155-178, BD Biosciences, Catalog No. 556652).RUC027WSF2024-223

[0369] Sometimes double-negative cells were sorted in the BD FACS Aria II and replated in U-bottom 96-well plates as described above for islet reclustering on the orbital shaker. After 24 hours, islets were dissociated into single cells for transduction of SIRPaE.140a, tCD47.140a, and CD64.140a (custom orders, Vectorbuilder) at a multiplicity of infection (MOI) of 5 and firefly luciferase lentivirus (custom order, GenTarget) at an MOI of 20. Spinfection was performed with the presence of protamine sulfate (10 pg / ml; Fresenius Kabi) at 300g for 15 min. Cells were replated in U-bottom 96-well plates as described above for islet reclustering on the orbital shaker. After 48 hours, B2M- / - CIITA- / - tCD47.140a+ CD64.140a+ islets were dissociated into single cells using ACCUMAX. They underwent cell sorting using an anti-CD47 antibody (clone B6H12, BD Biosciences) or an IgGl isotype- matched control antibody (clone MOPC-21, BD Biosciences) for tCD47.140a and PE antihuman CD64 (cat.no. 305008, Biolegend) or IgGl isotype-matched control antibody (clone MOPC-21, BD Biosciences) for CD64.140a. Alternatively, B2M- / - CIITA- / - SIRPaE.140a+ CD64.140a+ islets were dissociated into single cells using ACCUMAX and underwent cell sorting using an AF-647 anti-rabbit G4S linker antibody (Cat.no.69782, Clone E7O2V, Cell Signaling) to detect the SIRPaE.140a and matching AF-647 isotype control rabbit IgG (Cat.no.02-6102, Thermo Fisher), and PE anti-human CD64 (cat.no. 305008, Biolegend) or IgGl isotype-matched control antibody (clone MOPC-21, BD Biosciences) for CD64.140a. Luciferase expression was confirmed by adding D-luciferin (Promega). Signals were quantified with Ami HT (Spectral Imaging) in maximum photons s-1 cm-2 sr-1. Islet cells were replated in U-bottom 96-well plates as described above for islet reclustering on the orbital shaker until transplantation.

[0370] Quantification of human insulin. One hundred islet clusters were plated in one 6-well plate and 2 ml of islet media with 5.8 mM glucose (PIM(S) media, Prodo). Supernatants were collected after 24 h and ELISA assays for human insulin (Thermo Fisher Catalog No. KAQ1251) were performed according to the manufacture’s protocol. Briefly, islet cell supernatant was incubated with an anti-insulin antibody followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody and a peroxidase substrate. A microplate reader with an absorbance of optical density (OD) 450 nm (Molecular Devices Model No. 10822-512) was used to measure the insulin concentration of the standards and study samples. Insulin levels were calculated to ulU per million cells.

[0371] Splenocyte and CDC killing in XCelligence. Splenocyte and CDC killing assays were performed on the XCelligence MP platform. Specialized 96-well E-platesRUC027WSF2024-223(ACEA BioSciences) were coated and 40,000 target islet cells were plated in 100 pl islet media. When the cell index reached 0.7, effector cells were added at an E:T ratio of 1 :1. Additionally, 50 pl of untreated, complement-containing serum, 1 : 1 mixed with normal human complement (Quidel Catalog No. Al 13), was added to the target cells together with splenocytes. As controls, cells were grown in media only. Data were standardized and analyzed with the RTCA software (ACEA).

[0372] Elispots. For uni-directional Elispot assays, recipient splenocytes were isolated from humanized mice 6 days after islet transplantation and used as responder cells. Donor islet cells were mitomycin-treated (50 pg / ml for 30 min) and used as stimulator cells. One hundred thousand stimulator cells were incubated with one million recipient responder splenocytes for 48 h and IFN-y spot frequencies were enumerated using an Elispot plate reader (AID Diagnostika GmbH). Elispots with splenocytes from mice that received no transplants are shown as background.

[0373] Donor-specific antibodies (DSA). Sera from recipient animals were decomplemented by heating to 56 degrees C for 30 min. Equal amounts of sera and cell suspensions (5 million per ml) were incubated for 45 minutes at 4 degrees C. Cells were labeled with FITC-conjugated goat anti-human IgM (Thermo Fisher, Catalog No. 31575) and mean fluorescence intensity was analyzed by flow cytometry (Attune, Thermo Fisher). Islet cells incubated with serum from mice that received no transplants are shown as background.

[0374] In vivo BLI imaging. Mice were monitored until day 29. D-luciferin firefly potassium salt (375 mg / kg, Biosynth, Catalog No. L-8220) was dissolved in sterile PBS (pH 7.4, Gibco, Invitrogen) and was injected intraperitoneally (250 pl) into anesthetized mice. Animals were imaged using the Ami HT (Spectral Instruments Imaging). ROI bioluminescence was quantified in units of maximum photons per second per centimeter square per steradian. The maximum signal from an ROI was measured using Aura software (Spectral Instruments Imaging).

[0375] Glucose monitoring. Glucose measurements were performed until day 28 after islet transplantation using a glucometer (AccuCheck, Roche). A glucose concentration above 200 mg / dl was considered diabetic. For glucose challenge, mice received 2 g / kg glucose solution (Thermo Fisher) intraperitoneally and glucose concentration was determined 30 min after glucose injection.RUC027WSF2024-223

[0376] Innate in vitro killing by XCelligence. NK cell and macrophage killing assays were performed on the XCelligence MP platform (ACEA BioSciences). Specialized 96-well E-plates (ACEA BioSciences) were coated and 40,000 target islet cells were plated in 100 pl Prodo PIM(S) (Prodo) media. After the Cell Index reached 0.7, effector cells were added at an effector cell to target cell (E:T) ratio of 1 : 1. NK cells were stimulated with 1 pg / ml human IL-2 (Peprotech Catalog No. 200-02). In some wells, a humanized anti-EGFR antibody (Cetuximab, ichorbio) was added at a concentration of 1 pg / ml. As control, cells were grown in media only. Data were standardized and analyzed with the RTCA software (ACEA).Flow cytometry. To assess HLA class I expression, single islet cells were dissociated and labeled with APC-conjugated anti-HLA-A,B,C antibody (clone G46 2.6, BD Biosciences Catalog No. 555555) or APC-conjugated IgGl isotype-matched control antibody (clone MOPC-21, BD Biosciences Catalog No. 554681). To assess HLA class II expression, cells were incubated with AF647-conjugated anti-HLA-DR,DP,DQ antibody (clone Tu39, BD Biosciences Catalog No. 563591) or AF647-conjugated IgG2a isotype-matched control antibody (clone G155-178, BD Biosciences Catalog No. 565357). To assess tCD47.140a expression, the PerCP-Cy5-conjugated anti-CD47 (clone B6H12, BD Biosciences Catalog No. 561261) or PerCP-Cy5-conjugated IgGl isotype-matched control antibody (clone MOPC-21, BD Biosciences Catalog No. 550795) was used. To assess SIRPaE.140a expression, an AF-647 anti-rabbit G4S linker antibody (Clone E7O2V, Cell Signaling Catalog No. 69782) or matching AF-647 isotype control rabbit IgG (Thermo Fisher Catalog No. 02-6102) was used. To assess CD64.140a expression, a PE anti-human CD64 (Biolegend Catalog No. 305008) or IgGl isotype-matched control antibody (clone MOPC-21, BD Biosciences Catalog No. 555749) was used. Some samples were stimulated with IFNg and TNFa (both Peprotech Catalog Nos. 300-02 and 300-01 A, respectively) for 24 h at a concentration of 100 ng / ml. Representative histograms are shown.Example 7: Pig pancreatic islet cells for xenogeneic (human) transplantation

[0377] Engineered pig pancreatic islet cells survived human immunity and provided reliable diabetes therapy. To overcome organ shortages for patients with end-stage cardiac, pulmonary, or kidney diseases, the invention provides pig organs for human transplantation without immunosuppression. While some progress has been made to reduce xenogeneic transplantation rejection (e.g. GGTA1 knockouts to eliminate aGal expression), xenogeneicRUC027WSF2024-223 organs such as those from pigs are still vigorously rejected. The invention provides hypoimmune-edited cells for xenotransplantation. Here, comprehensively immune protected (CIP) pig pancreatic islet cells for human xenotransplantation are exemplified.

[0378] Wild type pig islets showed retained SLAs. The CIP engineering started with knockout of both the B2m and Ciita genes to deplete swine leukocyte antigen (SLA) class I and class II, which prevented T cell activation. The transgene tCD47.140a was expressed to inhibit human NK cells and macrophages. CD64.140a was expressed to protect the cells from human IgG antibody attack. The human CD99 and CD200 proteins were also expressed.They interact with inhibitory receptors on human PMNs to prevent PMN-mediated killing of the pig xenograft cells.

[0379] Pig WT islet cells (ICs) were susceptible to multiple immune attacks. While they were not killed by human IL-2 stimulated NK cells or macrophages, they were killed by human T cells primed against the pig WT ICs (Figure 29A). Pig WT ICs were also killed through cetuximab (anti-EGFR IgGl)-mediated killing in serum (CDC assay) and with human NK cells (ADCC assay, Figure 29B). Pig WT ICs were killed by human PMNs whether unstimulated or stimulated with DAMPs or IL-2 (Figure 29C).

[0380] Pig DKO (B2m-I- and Ciita- / -) ICs escaped primed human T cells but were killed by human IL-2 stimulated NK cells and macrophages (Figure 30A), through cetuximab CDC and ADCC (Figure 30B), as well as by stimulated and unstimulated human PMNs (Figure 30C).

[0381] Pig DKO tCD47.140a+ CD64.140a+ ICs were protected from primed human T cells, human IL-2 stimulated NK cells, and macrophages (Figure 31A). The pig DKO tCD47.140a+ CD64.140a+ ICs were also protected from both CDC and ADCC killing (Figure 31B). Human PMNs, however, whether unstimulated or stimulated, killed the pig DKO tCD47.140a+ CD64.140a+ ICs (Figure 31C).

[0382] Pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 ICs were protected against all human immune mechanisms including primed human T cells, human IL-2-stimulated NK cells, macrophages (Figure 32A); cetuximab-mediated CDC and ADCC killing (Figure 32B); and human PMNs, whether unstimulated or stimulated (Figure 32C).

[0383] Next, the CD99 and CD200 were exchanged for the chimeras CD99.140a and CD200.140a. Figure 32D shows pig DKO tCD47.140a+ CD64.140a+ CD99.140a+CD200.140a+ ICs incubated with primed human T cells, human IL-2-stimulated NK cells,RUC027WSF2024-223 and macrophages. Pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs were protected from primed T cells, NK cells, and macrophages. Figure 32E shows pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs incubated with 1 pg / ml cetuximab and either human serum or human NK cells. The pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs were protected from both CDC and ADCC killing. Figure 32F shows pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs incubated with human PMNs. The PMNs were either unstimulated or stimulated with cell debris (DAMPs) or human IL-2. Pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs were protected from human PMNs both unstimulated and stimulated. The pig target cells were transduced to express firefly luciferase, and their bioluminescence signal (BLI signal) was recorded over time. The relative BLI signal after 24 hours is shown.

[0384] Transplanted wild-type pig islets were killed in diabetic, xenogeneic humanized mice. The 500 WT pig islets were mixed with one million human PMNs and were injected into the hindlimb muscle. Islets were transduced to express firefly luciferase. Their survival was monitored through BLI (upper graph). All pig WT islet grafts were rejected within 10 days as shown by the drop of the BLI signal into the background (dashed line). Islets were injected 6 days after the mice received Streptozotocin (STZ) and all animals showed fasting blood glucose levels > 400 mg / dL (lower graph). The islet transplantation did not affect fasting blood glucose levels. All animals remained diabetic. (Figure 33A)

[0385] Transplanted pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 islets survived in diabetic, xenogeneic humanized mice. The assays were the same as those described for Figure 33 A. All pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 islet grafts survived and the increase in BLI signal indicates an increase in metabolic activity over time. This suggested improved vascularization. Islets were injected 6 days after the mice received STZ and all animals showed fasting blood glucose levels > 400 mg / dL (lower graph). In all humanized mice, pig DKO tCD47.140a+ CD64.140a+ CD99 CD200 islet transplantation lowered fasting blood glucose levels. All animals leveled out at around 200 mg / dl after 14 days and remained stable for at least about 50 days. (Figure 33B)

[0386] In Figure 33C, the CD99 and CD200 were exchanged for the chimeras CD99.140a and CD200.140a. The experiments were performed in the exact same way as for Figure 33B, just with pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ ICs. Five hundred thousand firefly luciferase-positive islet cells were mixed with one million human PMNs and were injected into the hindlimb muscle. All pig DKO tCD47.140a+RUC027WSF2024-223CD64.140a+ CD99.140a+ CD200.140a+ islet grafts survived for at least about 50 days post transplantation (upper graph). Islets were injected 6 days after the mice received STZ and all animals showed fasting blood glucose levels > 400 mg / dL (lower graph). In all mice, pig DKO tCD47.140a+ CD64.140a+ CD99.140a+ CD200.140a+ islet transplantation normalized fasting blood glucose levels by about 10 days post-transplantation and remained normalized at about 200 mg / dl and remained stable for at least about 50 days.Example 8: Hypoimmunogenic human parathyroid cells for allogeneic transplantation

[0387] Based on the surprising success of CIP pancreatic islet cells, other cell types were exemplified. Human parathyroid cells (Accegen, Fairfield, NJ, Cat. No. ABC-TC110L) were edited as outlined above for human primary beta cells. First, they were engineered to express firefly luciferase. DKO PTH cells were generated with knockouts of the B2M and CIITA genes. These PTH cells were then modified to express tCD47.140a (SEQ ID NO: 23), and CD64.140a (SEQ ID NO:24) transgenes.

[0388] Human WT, DKO, and CIP PTH cells were analyzed in vitro. WT PTH cells are killed by allogeneic, primed human T cells in culture but are protected from NK cell and macrophage killing (Figure 34A). WT PTH cells are susceptible to antibody-mediated killing via CDC or ADCC (Figure 34B). DKO PTH cells were not attacked by allogeneic, primed human T cells, but got killed by NK cells and macrophages (Figure 35A). DKO PTH cells were also susceptible to antibody-mediated killing via CDC or ADCC (Figure 35B). CIP PTH cells were fully protected from allogeneic, primed T cells as well as NK cells and macrophages (Figure 36A) and were protected from antibody-mediated killing (Figure 36B).

[0389] CIP PTH cells survived following transplantation into allogeneic humanized mice. A total of 250 thousand firefly luciferase-expressing WT or CIP PTH cells were transplanted into the hindlimb muscle. The survival was followed by BLI imaging as outlined for islet cell transplantation above. The WT PTH grafts vanished over 7-10 days. (Figure 37A). In contrast, the CIP PTH grafts showed stable BLI signals throughout the study period indicating graft survival. (Figure 37B).Example 9: Modified SIRPa engagers with inert intracellular domains (ICDs)

[0390] SIRPa engagers comprising one of two truncated ICDs with no signaling (SEQ ID NOs:38 -41) were expressed from lentiviruses using the EFla promoter. HumanRUC027WSF2024-223 primary islet cells were edited to deactivate B2M and CIITA (DKO) and expressed one of the original SIRPa engager (without an ICD) or the SIRPa engagers with truncated ICDs.

[0391] Human primary islet cells DKO tCD47.140a, DKO tCD47.140a.ml (SEQ ID NO: 40), or DKO tCD47.140a.ms (SEQ ID NO: 41) were generated and re-clustered. A total of 450 firefly luciferase-positive islet clusters were injected intramuscularly into diabetic, allogeneic humanized mice. Figure 38A shows that DKO tCD47.140a islet grafts survived for at least about 30 days in allogeneic humanized mice and alleviated diabetes. Figure 38B shows similar survival and efficacy for DKO tCD47.140a.ini islet grafts. Figure 38C shows similar survival and efficacy for DKO tCD47.140a. ms islet grafts.Example 10: Neutralized SIRPa kills grafted hypoimmune cellsNeutralizing SIRPa, blocking the SIRPa engager (on engineered cell)-SIRPa (on immune cell) interaction, or inhibiting the downstream SIRPa pathway provides a safety mechanism for the killing grafted hypoimmune cells that engage SIRPa. There are different strategies to block SIRPa signaling in immune cells and thus conditioning them to kill hypoimmune cells that depend on an active SIRPa pathway: (a) Antibodies: Antibodies binding SIRPa in a way that prohibits it from binding a binding partner and / or being activated to send a downstream signal in the immune cell, (b): Aptamers (DNA / RNA): Nucleic-acid ligands that bind a SIRPa engager (e.g. CD47) and block its interaction with SIRPa. (c): Peptides & peptidomimetics: Short sequences engineered to interfere with ligand binding to SIRPa. (d): Small molecules: small molecules interfering with SIRPa binding, (e): Bispecifics / multispecifics (antibody-based formats): Molecules that block binding of SIRPa. (f): Engineered SIRPa decoys: Non-signaling SIRPa ectodomains (often Fc-fused). (g): Gene- silencing / editing of SIRPa (in immune cells) through SIRPA knockout or RNAi / CRISPRi / ASO to reduce SIRPa expression in target cells, (h): Downstream signaling blockade: Inhibiting phosphatases recruited by SIRPa’ s ITIMs — especially SHP-l / SHP-2 — to blunt the inhibitory signal, (i): Targeted delivery / nanomedicine: Nanoparticles that deliver SIRPa-Fc or nucleic acids (e.g., siRNA / CRISPR) to locally disable the axis while limiting systemic exposure.

[0392] First, unedited (WT) primary human islets were analyzed in XCelligence cytotoxicity in vitro assays (Figure 39). Human WT islets were not susceptible to NK cell killing given their HLA expression. Additionally, WT islets were not killed by human NKRUC027WSF2024-223 cells combined with anti-SIRPa antibodies (aSIRPa) in ADCC assays. This showed that unedited cells (i.e. innate host cells) were not at risk from this safety strategy.

[0393] DKO SIRPaE.140a CD64.140a primary human islets, however, were reliably and quickly killed in ADCC assays with aSIRPa (Figure 40). While the engineered DKO SIRPaE.140a CD64.140a islets were not attacked by IL-2-stimulated human NK cells, they were quickly killed when aSIRPa was added. Higher concentrations led to faster killing.

[0394] Additionally, DKO tCD47.140a CD64.140a primary human islets were not susceptible to NK cell killing but were killed when a human aSIRPa was added to the NK cells (Figure 41). Higher doses again led to faster killing.

[0395] The aSIRPa safety strategy was confirmed in vivo. Firefly luciferasepositive DKO SIRPaE.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice (Figure 42). On day 0, one million human NK cells and 1 mg aSIRPa was administered (see arrows) and two more doses of 1 mg aSIRPa were given on days 1 and 2. The islet graft vanished (upper panel), and all mice remained diabetic (lower panel).

[0396] Similarly, firefly luciferase-positive DKO tCD47.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice (Figure 43). Six days before the transplantation (day -6), mice received STZ and became diabetic by day 0. On day 0, human NK cells and aSIRPa was administered (see arrows) and two more doses of 1 mg aSIRPa were given on days 1 and 2. The islet graft vanished (upper panel), and all mice remained diabetic (lower panel).

[0397] After confirmation that aSIRPa administration prevented freshly transplanted engineered islet engraftment, aSIRPa destruction of already engrafted islets was shown.(Figure 44). Firefly luciferase-positive DKO tCD47.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice. Six days before the transplantation (day -6), mice received STZ and became diabetic by day 0. On day 7, human NK cells and aSIRPa was administered (see arrows). Two more doses of 1 mg aSIRPa were given on days 8 and 9. The islet cells initially engrafted but vanished after day 7 (upper panel). Although fasting glucose levels were initially decreasing, all mice became diabetic again after day 7 (lower panel).RUC027WSF2024-223

[0398] This safety strategy is a general mechanism to kill transplanted therapeutic cells that rely, at least in part, on SIRPa engagement. Although the engineered islets discussed above utilize additional edits to comprehensively survive multiple human immune attacks, they rely on a functional SIRPa immune checkpoint in human innate immune cells. Thus, the aSIRPa safety strategy was shown to also work with engineered pig islets. This was first shown in vitro (Figure 45). Unedited (WT), primary pig islets were plated on the XCelligence platform and cytotoxicity assays were performed. WT primary pig islets were not susceptible to human IL-2 stimulated NK cell killing or ADCC using escalating doses of a human aSIRPa.

[0399] DKO tCD47.140a CD64.140a primary pig islets were also not susceptible to IL-2 stimulated NK cell killing but were killed when a human aSIRPa was added to the NK cells (Figure 46). Higher doses led to faster killing.

[0400] Similarly, DKO tCD47.140a CD64.140a CD99 CD200 primary pig islets were not susceptible to IL-2 stimulated NK cell killing but were killed when a human aSIRPa was added to the NK cells (Figure 47). Higher doses led to faster killing.

[0401] The aSIRPa antibodies killed the engineered pig islets in vivo. They were transplanted into diabetic, xenogeneic humanized mice (Figure 48). Here, destruction of the islet grafts was triggered even later than in the previous allotransplant analyses. After 30 days, robust engraftment of pig islets had been shown before with restored euglycemia in all mice. The late destruction after 30 days was shown.

[0402] Firefly luciferase-positive DKO tCD47.140a CD64.140a CD99 CD200 primary pig islets were transplanted into diabetic, allogeneic humanized mice additionally receiving PMNs with the graft on day 0. On day 30, human NK cells and aSIRPa were administered (see arrows) and two more doses of aSIRPa were given on days 31 and 32. The islet grafts, which had been stable until day 30, vanished (upper panel). All mice, which had reached fasting glucose levels of around 200 mg / dl, became diabetic again (lower panel).

[0403] Adoptive transfer of human SIRPa- / - NK cells, which lack a functioning SIRPa pathway, is an alternative safety strategy (Figure 49). This was first performed in the allotransplant setting with human islet grafts. Firefly luciferase-positive DKO tCD47.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice. On day 0, human SIRPa- / - NK cells (see W02020 / 263880, incorporated by reference hereinRUC027WSF2024-223 in its entirety) were administered (see arrows). The islet graft vanished (upper panel), and all mice remained diabetic (lower panel).

[0404] Additionally, firefly luciferase-positive DKO tCD47.140a CD64.140a primary human islets were transplanted into diabetic, allogeneic humanized mice. On day 7, human SIRPa- / - NK cells were administered (see arrows, Figure 50). These islet grafts, which had already engrafted, vanished (upper panel). All mice became diabetic again (lower panel).

[0405] The utility of human SIRPa- / - NK cells to destroy engineered pig islets was shown (Figure 51). Firefly luciferase-positive DKO tCD47.140a CD64.140a CD99 CD200 primary pig islets were transplanted into diabetic, allogeneic humanized mice additionally receiving PMNs with the graft on day 0. On day 30, human SIRPa- / - NK cells were administered (see arrows). The islet grafts, which had been stable until day 30, vanished (upper panel), and all mice, which had reached fasting glucose levels of around 200 mg / dl, became diabetic again (lower panel).

[0406] These data confirmed the utility and reliability of both the aSIRPa and the human SIRPa- / - NK cell safety strategies for engineered human and pig islets at early and late time points after transplantation.Methods

[0407] Cytotoxicity assays on the XCelligence platform. In vitro killing assays were performed on the XCelligence SP platform and MP platform (ACEA BioSciences, San Diego, CA). Special 96-well E-plates (ACEA BioSciences) were coated with collagen (Sigma- Aldrich) and 40,000 islet cells were plated in 100 pl medium. After the cell index (CI) reached 0.7, the effector cells with or without antibody were added. NK cells had been treated with 1 mg / ml human IL-2 for 24 h before the assay and continued in the assay. Data were standardized and analyzed with the RTCA software (ACEA BioSciences). An aSIRPa (MedChem Express, cat.no. HY-P990033) was used in the concentrations provided.

[0408] Transduction to express firefly luciferase. One hundred thousand islet cells were plated in one six-well plates and incubated overnight at 37 °C at 5% CO2. The next day, media was changed and one vial of Flue lentiviral particles expressing luciferase II gene under a re-engineered EFla promotor (GenTarget) was added to 1.5 ml media. After 36 h, 1 ml of cell media was added. After further 24 h, complete media change was performed. After 2 days, luciferase expression was confirmed by adding D-luciferin (Promega).RUC027WSF2024-223

[0409] Islet transplantation and killing. Humanized mice received intraperitoneal Streptozotocin (STZ) injections (60 mg / kg; Sigma-Aldrich Cat. No. 572201) 6 days before islet transplantation. All animals showed fasting blood glucose levels > 400 mg / dL at the time of transplant. Either 450 human or 500 pig islets were transplanted. Pig islets were mixed with one million human PMNs and were injected into the hindlimb muscle. No PMNs were injected for human islet transplants. Some mice received 1 mg aSIRPa antibody (MedChem Express, Princeton, NJ, Cat. No. HY-P990033) with one million human NK cells at the time point indicated. Some mice received one million SIRPa- / - NK cells at the time point indicated.Example 11: Islet cell transplantation in single cell suspension

[0410] The invention provides off-the-shelf islet cell therapies using single cell suspensions. This eliminates the need for islet re-clustering at GMP facilities that can take up to about 7 days.

[0411] Firefly luciferase-positive human DKO tCD47.140a CD64.140a islet clusters were dissociated in single cells using AccuMax (StemCell Technologies) for 10 min at 37°C. Cells were spun down (300g, 4 min, room temperature). For in vivo analyses, 450,000 cells were either resuspended in 60 l saline (Gibco) or were allowed to re-cluster on an orbital shaker for 24 hours. The clusters were then carefully resuspended in 60 l saline. Animals received either the islet single cell suspensions or the re-clustered product. Injections were performed using 27G needles (Braun) and 1ml syringes (no dead space syringe; Braun). Islet survival was followed by BLI imaging and islet cell function was assessed by serial fasting blood glucose monitoring.

[0412] Figure 52A shows the BLI imaging graphs for the survival of human DKO tCD47.140a CD64.140a islet cells after they were transplanted in single cell suspension (top) or after re-clustering (bottom) into diabetic NSG mice in a first analytical run (Runl). No differences in survival were observed.

[0413] Figure 52B shows the function of the above transplanted islet cells to alleviate diabetes in diabetic NSG mice. Six days before the injection of islet cells, mice received intraperitoneal Streptozotocin (STZ) injections (60 mg / kg; Sigma-Aldrich Cat. No. 572201). Blood was regularly drawn before and after transplantation and blood glucose was measured. The islet cells that were transplanted in single cell suspension are shown on top and thoseRUC027WSF2024-223 transplanted after re-clustering are shown on the bottom. Both groups were similarly effective in alleviating diabetes and no differences between groups were seen.

[0414] Figure 53A shows the BLI imaging graphs for the survival of human DKO tCD47.140a CD64.140a islet after they were transplanted in single cell suspension (top) or after re-clustering (bottom) into diabetic NSG mice in a second analytical run (Run 2). No differences in survival were observed. The cells survived for at least about 20 days after transplantation.

[0415] Figure 53B shows the function of the above transplanted islet cells to alleviate diabetes in diabetic NSG mice. Six days before the injection of islet cells, mice received intraperitoneal Streptozotocin (STZ) injections (60 mg / kg). Blood was regularly drawn before and after transplantation and blood glucose was measured. The islet cells that were transplanted in single cell suspension are shown on top and those transplanted after reclustering are shown on the bottom. Both groups were similarly effective in alleviating diabetes and no differences between groups were seen.

[0416] After 28 days, the animals were euthanized and injection sites were cut out and processed for histology. Figure 54A shows a representative picture of an islet cluster that formed spontaneously after the injection of single cells in suspension in Run 1. The clusters were approximately 100 pm in diameter. Figure 54B shows a representative picture of an islet cluster that formed spontaneously after the injection of single cells in suspension in Run 2. The spontaneous islet clusters were again approximately 100 pm in diameter.

[0417] Primary human islets were heterogeneous, typically -120-150 pm in diameter. The slightly smaller size of spontaneous clusters that formed in vivo after the injection of single cell suspensions helps resist ischemic injury during transplantation. The spontaneous clustering also avoids the need for prior re-clustering in vitro. Thus, the invention provides a much faster and easier method for treating diabetes that comprises injecting single islet cell suspensions into subjects. This efficient method is far less cumbersome than the prior art.Example 12: Islet transplantation into humans

[0418] Comprehensively Immune Protected islets or islet cells (either human or if xenogeneic then additionally expressing a CD200R engager and a CD99 ligand engager) are transplanted into human diabetic patients without immunosuppression. Approximately 5000 -RUC027WSF2024-22312000 islet equivalents per kilogram of body weight are suspended and injected intramuscularly, into the portal vein, or into another vascularized location of the body.

[0419] The islets for intraportal infusion are suspended in CMRL- 1066-based “transplant media” (Mediatech). In some embodiments, it is supplemented with human serum albumin (—2—5%) and heparin. In other embodiments, an antibiotic is also added. In other embodiments, Hanks’ balanced salt solution with human albumin is used. The final product may be packaged in Ricordi infusion bags at about a 200-400 mL total volume. For intramuscular injection, islets are suspended in much smaller volumes of media, about 3 - 25 mL total volume.

[0420] Patients are monitored for insulin independence over a period of months to years. Insulin independence is defined herein as freedom from the need to take exogenous insulin, with adequate glycemic control. This is defined by a glycated hemoglobin level of less than 6.5% with a glucose level after an overnight fast not exceeding 140 mg per deciliter (7.8 mmol per liter) more than three times in any week (based on the morning fasting glucose level) and not exceeding 2-hour postprandial levels of 180 mg per deciliter (10 mmol per liter) more than four times per week. See Carlsson et aL, N Engl J Med. 2025 Aug 4. doi: 10.1056 / NEJMoa2503822; Reichman et al., VX-880-101 N Engl J Med. 2025 Jun 20. doi: 10.1056 / NEJMoa2506549; and Shapiro et al., NEnglJMed. 2006 Sep 28;355(13): 1318-30. The foregoing are incorporated by reference herein in their entirety.

[0421] IX. Exemplary sequences:SEQ ID NO:1 - Anti-CD200R Variable Heavy Chain (VH) DomainQVQLQESGPGLVKPSETLSLTCTVSGYTITSGYDWSWIRQPPGKGLEWIGYINYGGSTNYKP SLGSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARYNEYKSYIYDWYFDFWGQGTLVTVSSSEQ ID NO:2 - Anti-CD200R Variable Light Chain (VL) DomainDIQMTQSPSSLSASVGDRVTITCKASKNIRSYLAWYQQKPGKAPKLLIYSGSTLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQHHEYPLTFGQGTKVEIKRSEQ ID NO:3 - CD8a signal domainMALPVTALLLPLALLLHAARPSEQ ID NO:4 - (GGGGS)3LinkerGGGGSGGGGSGGGGSSEQ ID NO:5 - CD8a hinge domainTTTPAPRPPTPAPTIASQPLSLRPEACRPAAAVHTRGLDFACDRUC027WSF2024-223SEQ ID NO: 6 - Platelet Derived Growth Factor Receptor (PDGFR or CD 140a) Transmembrane Domain (TMD)AAVLVLLVIVI ISLIVLWIWSEQ ID NO:7 - Exemplary CD200R Engager Molecule (CD200RE.140a)MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSETLSLTCTVSGYTITSGYDWSWIRQP PGKGLEWIGYINYGGSTNYKPSLGSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARYNEYK SYIYDWYFDFWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAS KNIRSYLAWYQQKPGKAPKLLIYSGSTLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQHHEYPLTFGQGTKVEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAAVHTRGLDFAC DAAVLVLLVIVI ISLIVLWIWSEQ ID NO:8 - Human fi-2-MicroglobulinMSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKN GERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDISEQ ID NO:9 - Human CIITA protein, 160 amino acid N-terminusMRCLAPRPAGSYLSEPQGSSQCATMELGPLEGGYLELLNSDADPLCLYHFYDQMDLAGEEEI ELYSEPDTDTINCDQFSRLLCDMEGDEETREAYANIAELDQYVFQDSQLEGLSKDI FKHIGP DEVIGESMEMPAEVGQKSQKRPFPEELPADLKHWKPSEQ ID NO: 10 - Human CD47MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKG RDI YTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGE TI IELKYRWSWFSPNENILIVI FPI FAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVI TVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIA Y I LAWGLS LG I AAC I PMHGPLL I S GLS I LALAQLLGLVYMKFVESEQ ID NO: 11 - Herpes Simplex Virus Thimidine Kinase (HSV-tk)MASYPCHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRLEQKMPTLLRVYIDGPHGMGK TTTTQLLVALGSRDDIVYVPEPMTYWQVLGASETIANIYTTQHRLDQGEISAGDAAWMTSA QITMGMPYAVTDAVLAPHVGGEAGSSHAPPPALTLI FDRHPIAALLCYPAARYLMGSMTPQA VLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRY LQGGGSWWEDWGQLSGTAVPPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAW ALDVLAKRLRPMHVFILDYDQSPAGCRDALLQLTSGMVQTHVTTPGS IPTICDLARTFAREM GEANSEQ ID NO:12 - Escherichia coli Cytosine Deaminase (EC-CD)MSNNALQTI INARLPGEEGLWQIHLQDGKISAIDAQSGVMPITENSLDAEQGLVIPPFVEPH IHLDTTQTAGQPNWNQSGTLFEGIERWAERKALLTHDDVKQRAWQTLKWQIANGIQHVRTHV DVSDATLTALKAMLEVKQEVAPWIDLQIVAFPQEGILSYPNGEALLEEALRLGADWGAIPH FE FTRE YGVE S LHKT FALAQKYDRL I DVHCDE I DDEQSRFVE TVAALAHHEGMGARVTASHT TAMHSYNGAYTSRLFRLLKMSGINFVANPLVNIHLQGRFDTYPKRRGITRVKEMLESGINVC FGHDDVFDPWYPLGTANMLQVLHMGLHVCQLMGYGQINDGLNLITHHSARTLNLQDYGIAAG NSANLI ILPAENGFDALRRQVPVRYSVRGGKVIASTQPAQTTVYLEQPEAIDYKRSEQ ID NO:13 - Truncated human Caspase 9GFGDVGALESLRGNADLAYILSMEPCGHCLI INNVNFCRESGLRTRTGSNIDCEKLRRRFSS LHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVWILSHGCQASHLQFPGAVYGTDGCPV SVEKIVNI FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQRUC027WSF2024-223EGLRTFDQLDAISSLPTPSDI FVSYSTFPGFVSWRDPKSGSWYVETLDDI FEQWAHSEDLQS LLLRVANAVSVKGI YKQMPGCFNFLRKKLFFKTSSEQ ID NO: 14 - Human CD64NM 000566MWFLTTLLLWVPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTA TQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRC HAWKDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVK ELFPAPVLNASVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQIL TARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHVLFYLAVGIMFLVNTVLW VTIRKELKRKKKWDLEISLDSGHEKKVISSLQEDRHLEEELKCQEQKEEQLQEGVHRKEPQG ATSEQ ID NO: 15 - Human CD52NM 001803MKRFLFLLLTISLLVMVQIQTGLSGQNDTSQTSSPSASSNISGGI FLFFVANAI IHLFCFSSEQ ID NO: 16 - Human CD16 FCGR3ANM 001803MAEGTLWQILCVSSDAQPQTFEGVKGADPPTLPPGSFLPGPVLWWGSLARLQTEKSDEVSRK GNWWVTEMGGGAGERLFTSSCLVGLVPLGLRISLVTCPLQCGIMWQLLLPTALLLLVSAGMR TEDLPKAWFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATV DDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQ NGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPP GYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDKSEQ ID NO: 17 - Human CD16 FCGR3BNM 001803MWQLLLPTALLLLVSAGMRTEDLPKAWFLEPQWYSVLEKDSVTLKCQGAYSPEDNSTQWFH NENLISSQASSYFIDAATVNDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPI HLRCHSWKNTALHKVTYLQNGKDRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKNVSSETV NITITQGLAVSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNISEQ ID NO: 18 - Human CD32 FCGR2ANM 001803MTMETQMSQNVCPRNLWLLQPLTVLLLLASADSQAAAPPKAVLKLEPPWINVLQEDSVTLTC QGARSPESDS IQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPVHLTVLSEWL VLQTPHLEFQEGETIMLRCHSWKDKPLVKVTFFQNGKSQKFSHLDPTFS IPQANHSHSGDYH CTGNIGYTLFSSKPVTITVQVPSMGSSSPMGI IVAWIATAVAAIVAAWALIYCRKKRISA NSTDPVKAAQFEPPGRQMIAIRKRQLEETNNDYETADGGYMTLNPRAPTDDDKNI YLTLPPN DHVNSNNSEQ ID NO: 19 - Human CD32 FCGR2BNM 001803MGILSFLPVLATESDWADCKSPQPWGHMLLWTAVLFLAPVAGTPAAPPKAVLKLEPQWINVL QEDSVTLTCRGTHSPESDS IQWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPV HLTVLSEWLVLQTPHLEFQEGETIVLRCHSWKDKPLVKVTFFQNGKSKKFSRSDPNFS IPQA NHSHSGDYHCTGNIGYTLYSSKPVTITVQAPSSSPMGI IVAWTGIAVAAIVAAWALI YCR KKRISALPGYPECREMGETLPEKPANPTNPDEADKVGAENTITYSLLMHPDALEEPDDQNRISEQ ID NO:20 - Human CD32 FCGR2CRUC027WSF2024-223NM 001803MGILSFLPVLATESDWADCKSPQPWGHMLLWTAVLFLAPVAGTPAAPPKAVLKLEPQWINVL QEDSVTLTCRGTHSPESDS IPWFHNGNLIPTHTQPSYRFKANNNDSGEYTCQTGQTSLSDPV HLTVLSEWLVLQTPHLEFQEGETIVLRCHSWKDKPLVKVTFFQNGKSKKFSRSDPNFS IPQA NHSHSGDYHCTGNIGYTLYSSKPVTITVQAPSSSPMGI IVAWTGIAVAAIVAAWALI YCRKKRISANSTDPVKAAQFEPPGRQMIAIRKRQPEETNNDYETADGGYMTLNPRAPTDDDKNIY LTLPPNDHVNSNNSEQ ID NO:21 - Human CD99NP 002405.1MARGAALALL LFGLLGVLVA APDGGFDLSD ALPDNENKKP TAIPKKPSAG DDFDLGDAW DGENDDPRPP NPPKPMPNPN PNHPSSSGSF SDADLADGVS GGEGKGGSDG GGSHRKEGEE ADAPGVIPGI VGAVWAVAG AISSFIAYQK KKLCFKENAE QGEVDMESHR NANAEPAVQR TLLEKSEQ ID NO:22 - Human CD200NP 001352780.1MERLVIRMPF SHLSTYSLVW VMAAWLCTA QVQWTQDER EQLYTPASLK CSLQNAQEAL IVTWQKKKAV SPENMVTFSE NHGWIQPAY KDKINITQLG LQNSTITFWN ITLEDEGCYM CLFNTFGFGK ISGTACLTVY VQPIVSLHYK FSEDHLNITC SATARPAPMV FWKVPRSGIE NSTVTLSHPN GTTSVTS ILH IKDPKNQVGK EVICQVLHLG TVTDFKQTVN KGYWFSVPLL LS IVSLVILL VLIS ILLYWK RHRNQDRGEL SQGVQKMTSEQ ID NO:23 - tCD47 (tCD47.140a chimera)MWPLVAALLL GSACCGSAQL LFNKTKSVEF TFCNDTWIP CFVTNMEAQN TTEVYVKWKF KGRDIYTFDG ALNKSTVPTD FSSAKIEVSQ LLKGDASLKM DKSDAVSHTG NYTCEVTELT REGETI IELK YRWSWFSPN EAAVLVLLVI VI ISLIVLW IWSEQ ID NO:24 - tCD64 (CD64.140a chimera)MWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPG SSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEI HRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWN SNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTS PLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTA RREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPVW FHAAVLVLLV IVI ISLIVLV VIWSEQ ID NO:25 - Swine Beta-2-microglobulinMAPLVALVLL GLLSLSGLDA VARPPKVQVY SRHPAENGKP NYLNCYVSGF HPPQIEIDLL KNGEKMNAEQ SDLSFSKDWS FYLLVHTEFT PNAVDQYSCR VKHVTLDKPK IVKWDRDHSEQ ID NO:2< - Swine CiitaMRCLAPRPAG SYLPEPQGSG QCATMELGPL EGGYLELLNS SADPLQLYHLYDRMDLAGEE EIELCSEPDT DTINCEQFSR LLCDMEADEE TRETYAS IAELDQYVFQDSQ LEGLGKDI FI EHIGLEEMIS ESVEVLEDSG RKSQKRSFPEELPADLKHRK LAEPLAMPMV TGTFLVGPVS DSSARPCPSP PALFNKESTPRUC027WSF2024-223SQAQLEDAVP MPAPPSGSLL SCLSVPAGPI QI IPTLSTLP QGLWHISGAG TGVSS ILIYQ GEMTQASQAP PVHSLPKSPD RPGSTSPFAP SAADLPSMPE PALTSRANMT EGSVSPTQCS GDQEASSRLP KWPETVEQFH HSLRDRYQAK PAGPEGILVE VDLVRVRLER SSSKSQEREL ASLDWAERQP ARGGLAEVLL AASDRQGPRE TQVIAVLGKA GQGKSHWAQA VSWAWADGQL PQYDFVFCIP CHCLDRPGNT YRLQDLLFSL GPQPLPMDDE VFSYILRRPD RVLLILDAFE EREAQDGFVH SAGGPLSSEP RSLRGLLAGL LQRKLLRGCT LLLTARPRGR LAQSLSKADA LFEVAGFSAQ QAKTYMLRYF ECRGARERQK RALELLQAQP FLLSHSHSPS VCRAVCRLSE TLLELGEEAE LPSTLTGLYV GLLGPAARES PPGALVGLAR LAWELGRRHH SSLQEGQFPS AEARAWAVAQ GLVQRAPGAP GAPELAFSSF LLQCFLGAVW LALSSEIKDK ELPQYLALTP RKKRPYDNWL EAVPRFLVGL VFQPRARCLG ALAGLVAATL ADRKQKVLNR YLKRLQPGTL QAGRLLELLH CTHEALDSGL WQHVLQGLPT QLSFLGTRLT PPDTHVLGSA LVAAGRDFSL DLRSTGIDPS GLGSLVGLSC VTHFRAALSD TVGLWESLQQ RGETKLLQAL EEKFTIEPFK AKSMKDVEDL GNLVQIQRTR SSSEDMAGEL PAVRDLKKLE FALGPVLGPQ AFPKLVRILE AFSSLQHLDL DSLSENKIGD EGVAQLSATF PQLKALETLN LSQNNISDVG ACQLAKALPS LAASLLRLSL YNNCICDVGA ESLAHVLPDM GSLRVLDVQY NKFTAAGAQQ LAASLRKCPH METLAMWTPT IPFGVQEHLQ QQDSRISSEQ ID NO:27 - SIRPaE.140a (Anti-SIRPa / CD140a TMD fusion)MALPVTALLL PLALLLHAAR PQVQLVESEG GLVQPGGSLR LSCAASGFTF SSYEMNWVRQ APGKGLEWVS YISSSGSTIY YADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARE AKGYYYGMDV WGQGTTVTVS SGGGGSGGGG SGGGGSQPVL TQSPSVSVSP GQTAS ITCSG DKLGDTYACW YQQKPGQSPV LVIYQDTKRP SGIPERFSGS NSGNTATLTI SGTQAMDEAD YYCQAWDSSTWFGGGTKLT VLTTTPAPRP PTPAPTIASQ PLSLRPEACR PAAAVHTRGL DFACDAAVLV LLVIVI ISLI VLWIWSEQ ID NO:2< - CD200.140a chimera MERLVIRMPF SHLSTYSLVW VMAAWLCTA QVQWTQDER EQLYTPASLK CSLQNAQEAL IVTWQKKKAV SPENMVTFSE NHGWIQPAY KDKINITQLG LQNSTITFWN ITLEDEGCYM CLFNTFGFGK ISGTACLTVY VQPIVSLHYK FSEDHLNITC SATARPAPMV FWKVPRSGIE NSTVTLSHPN GTTSVTS ILH IKDPKNQVGK EVICQVLHLG TVTDFKQTVN KGAAVLVLLV IVI ISLIVLV VIWSEQ ID NO:29 - CD200 Extracellular domainQVQWTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGWIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTS ILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGSEQ ID NQ:30 - CD99.140a chimeraMARGAALALL LFGLLGVLVA APDGGFDLSD ALPDNENKKP TAIPKKPSAG DDFDLGDAW DGENDDPRPP NPPKPMPNPN PNHPSSSGSF SDADLADGVS GGEGKGGSDG GGSHRKEGEE ADAAVLVLLV IVI ISLIVLV VIWSEQ ID NO:31 - CD99 Extracellular domainRUC027WSF2024-223DGGFDLSDALPDNENKKPTAIPKKPSAGDDFDLGDAWDGENDDPRPPNPPKPMPNPNPNHP SSSGSFSDADLADGVSGGEGKGGSDGGGSHRKEGEEADSEQ ID NO:32 - HUMAN Thrombomodulin (TRBM)MLGVLVLGALALAGLGFPAPAEPQPGGSQCVEHDCFALYPGPATFLNASQICDGLRGHLM TVRSSVAADVISLLLNGDGGVGRRRLWIGLQLPPGCGDPKRLGPLRGFQWVTGDNNTSYS RWARLDLNGAPLCGPLCVAVSAAEATVPSEPIWEEQQCEVKADGFLCEFHFPATCRPLAV E P GAAAAAVS I T YG T P FAARGAD FQAL P VG S S AAVAP L GL QLMC T AP P GAVQGHWARE AP GAWDCSVENGGCEHACNAIPGAPRCQCPAGAALQADGRSCTASATQSCNDLCEHFCVPNP DQPGSYSCMCETGYRLAADQHRCEDVDDCILEPSPCPQRCVNTQGGFECHCYPNYDLVDG ECVEPVDPCFRANCEYQCQPLNQTSYLCVCAEGFAPIPHEPHRCQMFCNQTACPADCDPN TQASCECPEGYILDDGFICTDIDECENGGFCSGVCHNLPGTFECICGPDSALARHIGTDCDSGKVDGGDSGSGEPPPSPTPGSTLTPPAVGLVHSGLLIGIS IASLCLWALLALLCHLR KKQGAARAKMEYKCAAPSKEWLQHVRTERTPQRLSEQ ID NO:33 - Human Membrane cofactor protein (MCP or CD46)MEPPGRRECPFPSWRFPGLLLAAMVLLLYSFSDACEEPPTFEAMELIGKPKPYYEIGERV DYKCKKGYFYIPPLATHTICDRNHTWLPVSDDACYRETCPYIRDPLNGQAVPANGTYEFG YQMHFICNEGYYLIGEEILYCELKGSVAIWSGKPPICEKVLCTPPPKIKNGKHTFSEVEV FEYLDAVTYSCDPAPGPDPFSLIGESTIYCGDNSVWSRAAPECKWKCRFPWENGKQIS GFGKKFYYKATVMFECDKGFYLDGSDTIVCDSNSTWDPPVPKCLKVLPPSSTKPPALSHS VSTSSTTKSPASSASGPRPTYKPPVSNYPGYPKPEEGILDSLDVWVIAVIVIAIWGVAV ICWPYRYLQRRKKKGTYLTDETHREVKFTSLSEQ ID NO:34 - Human Complement decay-accelerating factor OSMTVARPSVPAALPLLGELPRLLLLVLLCLPAVWGDCGLPPDVPNAQPALEGRTSFPEDTV ITYKCEESFVKIPGEKDSVICLKGSQWSDIEEFCNRSCEVPTRLNSASLKQPYITQNYFP VGTWEYECRPGYRREPSLSPKLTCLQNLKWSTAVEFCKKKSCPNPGEIRNGQIDVPGGI LFGATISFSCNTGYKLFGSTSSFCLISGSSVQWSDPLPECREIYCPAPPQIDNGI IQGER DHYGYRQSVTYACNKGFTMIGEHS I YCTVNNDEGEWSGPPPECRGKSLTSKVPPTVQKPT TVNVPTTEVSPTSQKTTTKTTTPNAQATRSTPVSRTTKHFHETTPNKGSGTTSGTTRLLS GHTCFTLTGLLGTLVTMGLLTSEQ ID NO:35 - Human Endothelial protein C receptor OS (EPCR)MLTTLLPILLLSGWAFCSQDASDGLQRLHMLQISYFRDPYHVWYQGNASLGGHLTHVLEG PDTNTTI IQLQPLQEPESWARTQSGLQSYLLQFHGLVRLVHQERTLAFPLTIRCFLGCEL PPEGSRAHVFFEVAVNGSSFVSFRPERALWQADTQVTSGWTFTLQQLNAYNRTRYELRE FLEDTCVQYVQKHISAENTKGSQTSRSYTSLVLGVLVGSFI IAGVAVGI FLCTGGRRCSEQ ID NO:36 - Human Heme oxygenase 1 OSMERPQPDSMPQDLSEALKEATKEVHTQAENAEFMRNFQKGQVTRDGFKLVMASLYHI YVA LEEEIERNKESPVFAPVYFPEELHRKAALEQDLAFWYGPRWQEVIPYTPAMQRYVKRLHE VGRTEPELLVAHAYTRYLGDLSGGQVLKKIAQKALDLPSSGEGLAFFTFPNIASATKFKQ LYRSRMNSLEMTPAVRQRVIEEAKTAFLLNIQLFEELQELLTHDTKDQSPSRAPGLRQRA SNKVQDSAPVETPRGKPPLNTRSQAPLLRWVLTLSFLVATVAVGLYAMSEQ ID NO:37 - Human CD59 glycoprotein OSMGIQGGSVLFGLLLVLAVFCHSGHSLQCYNCPNPTADCKTAVNCSSDFDACLITKAGLQV YNKCWKFEHCNFNDVTTRLRENELTYYCCKKDLCNFNEQLENGGTSLSEKTVLLLVTPFL AAAWSLHPRUC027WSF2024-223SEQ ID NO:38 - SIRPotE.140a.mlMALPVTALLL PLALLLHAAR PQVQLVESEG GLVQPGGSLR LSCAASGFTF SSYEMNWVRQ APGKGLEWVS YISSSGSTIY YADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARE AKGYYYGMDV WGQGT TVTVS SGGGGSGGGG SGGGGSQPVL TQSPSVSVSP GQTAS ITCSG DKLGDTYACW YQQKPGQSPV LVIYQDTKRP SGIPERFSGS NSGNTATLTI SGTQAMDEAD YYCQAWDSST WFGGGTKLT VLTTTPAPRP PTPAPTIASQ PLSLRPEACR PAAAVHTRGL DFACDAAVLV LLVIVI ISLI VLWIWYQEEELCFEESEQ ID NO:39 - SIRPocE.140a.msMALPVTALLL PLALLLHAAR PQVQLVESEG GLVQPGGSLR LSCAASGFTF SSYEMNWVRQ APGKGLEWVS YISSSGSTIY YADSVKGRFT ISRDNAKNSL YLQMNSLRAE DTAVYYCARE AKGYYYGMDV WGQGT TVTVS SGGGGSGGGG SGGGGSQPVL TQSPSVSVSP GQTAS ITCSG DKLGDTYACW YQQKPGQSPV LVIYQDTKRP SGIPERFSGS NSGNTATLTI SGTQAMDEAD YYCQAWDSST WFGGGTKLT VLTTTPAPRP PTPAPTIASQ PLSLRPEACR PAAAVHTRGL DFACDAAVLV LLVIVI ISLI VLWIWYQSEQ ID NQ:40 - tCD47.140a.iniMWPLVAALLL GSACCGSAQL LFNKTKSVEF TFCNDTWIP CFVTNMEAQN TTEVYVKWKF KGRDIYTFDG ALNKSTVPTD FSSAKIEVSQ LLKGDASLKM DKSDAVSHTG NYTCEVTELT REGETI IELK YRWSWFSPN EAAVLVLLVI VI ISLIVLW IWYQEEELCF EESEQ ID NO:41 - tCD47.140a.msMWPLVAALLL GSACCGSAQL LFNKTKSVEF TFCNDTWIP CFVTNMEAQN TTEVYVKWKF KGRDIYTFDG ALNKSTVPTD FSSAKIEVSQ LLKGDASLKM DKSDAVSHTG NYTCEVTELT REGETI IELK YRWSWFSPN EAAVLVLLVI VI ISLIVLW IWYQSEQ ID NO: 42 - Synthetic Mutated Long ICD (ml)YQEEELCFEESEQ ID NO:43 - Synthetic Mutated short ICD (ms)YQSEQ ID NO:44 - CD47 Extra-Cellular DomainMWPLVAALLL GSACCGSAQL LFNKTKSVEF TFCNDTWIP CFVTNMEAQN TTEVYVKWKF KGRDIYTFDG ALNKSTVPTD FSSAKIEVSQ LLKGDASLKM DKSDAVSHTG NYTCEVTELT REGETI IELK YRWSWFSPN ESEQ ID NO:45 - CD64 Extra-Cellular DomainMWFLTTLLLW VPVDGQVDTT KAVITLQPPW VSVFQEETVT LHCEVLHLPGSSSTQWFLNG TATQTSTPSY RITSASVNDS GEYRCQRGLS GRSDPIQLEIHRGWLLLQVS SRVFTEGEPL ALRCHAWKDK LVYNVLYYRN GKAFKFFHWNSNLTILKTNI SHNGTYHCSG MGKHRYTSAG ISVTVKELFP APVLNASVTSPLLEGNLVTL SCETKLLLQR PGLQLYFSFY MGSKTLRGRN TSSEYQILTARREDSGLYWC EAATEDGNVL KRSPELELQV LGLQLPTPVW EHRUC027WSF2024-223SEQ ID NO:46 - Anti-SIRPa AntibodyDIQMTQSPSS VSASVGDRVT ITCRASQGIS SWLAWYQQKP GKAPKLLIYA ASNLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ GASFPITFGG GTKVEIKRTV AAPSVFI FPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGECQVQLVQ SGAEVKKPGA SVKVSCKASG YTFRGYGISW VRQAPGQGLE WMGWISAYGG ETNYAQKLQG RVTMTTDTST STAYMELRSL RSDDTAVYYC AREAGSSWYD FDLWGRGTLV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SWTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCW VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRW SVLTVLHQDW LNGKEYKCAV SNKALPAPIE KTISKARGQP REPQVYTLPP SREEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

[0422] All publications and patent documents disclosed or referred to herein are incorporated by reference in their entirety. The foregoing description has been presented only for purposes of illustration and description. This description is not intended to limit the invention to the precise form disclosed. It is intended that the scope of the invention be defined by the claims appended hereto.

Claims

RUC027WSF2024-223CLAIMSWhat is claimed:

1. A cell, comprising a reduced or eliminated surface leukocyte antigen class I, a first cellsurface molecule that is a SIRPa engager molecule (SIRPaE) expressed on a cell surface membrane of said cell by a first transmembrane domain, wherein said first cell-surface molecule does not comprise a domain that induces intracellular signaling, and a second cell-surface molecule that sequesters antibodies expressed on said cell surface membrane comprising a CD64 extracellular domain bound to said cell surface by a second transmembrane domain.

2. The cell of claim 1, wherein either said first or said second transmembrane domain, or both, comprise at least a 90% sequence identity to SEQ ID NO:6.

3. The cell of claim 1, wherein either said first or said second transmembrane domain, or both, comprise the sequence of SEQ ID NO:6.

4. The cell of any one of claims 1-3, wherein said SIRPaE has a CD47 extra-cellular domain having at least a 90% sequence identity to SEQ ID NO:44 and a transmembrane domain having at least a 90% sequence identity to SEQ ID NO:6.

5. The cell of claim 4, wherein said CD47 extra-cellular domain has the sequence of SEQ ID NO:44 and said transmembrane domain has the sequence of SEQ ID NO:6.

6. The cell of claim 1, wherein said SIRPaE has at least a 90% sequence identity to SEQ ID NO:27.

7. The cell of claim 6, wherein said SIRPaE has the sequence of SEQ ID NO:27.

8. The cell of claim 1, wherein said SIRPaE has at least a 90% sequence identity to any one of SEQ ID NOS:23, 38, 39, 40, or 41.

9. The cell of claim 8, wherein said SIRPaE has the sequence of any one of SEQ ID NOS:23, 38, 39, 40, or 41.

10. The cell of any one of claims 1-9, wherein said CD64 extracellular domain has at least a 90% sequence identity to SEQ ID NO:45.

11. The cell of claim 10, wherein said CD64 extracellular domain has the sequence of SEQ ID NO:45.

12. The cell of claim 1, wherein said antibody sequestration molecule has CD64 activity and has at least a 90% sequence identity to SEQ ID NO:24.

13. The cell of claim 12, wherein said antibody sequestration molecule has the sequence of SEQ ID NO:24.RUC027WSF2024-22314. The cell of claim 1, wherein said antibody sequestration molecule has CD64 activity and has at least a 90% sequence identity to SEQ ID NO: 14.

15. The cell of claim 14, wherein said antibody sequestration molecule has the sequence of SEQ ID NO: 14.

16. The cell of any one of claims 1-15, further comprising a third cell surface molecule, wherein said third cell-surface molecule is a CD200R engager molecule, wherein said CD200R engager molecule has a CD200R engaging domain that engages a CD200R on immune cells following transplantation of said cell into a subject.

17. The cell of claim 16, wherein said third cell surface molecule is a chimera.

18. The cell of either one of claims 16 or 17, wherein said CD200R engaging domain comprises an immunoglobulin heavy chain domain.

19. The cell of any one of claims 16 or 17, wherein said third cell surface molecule lacks a CD200 intracellular signaling function.

20. The cell of claim 18, wherein said immunoglobulin heavy chain domain is a variable heavy chain domain having at least a 90% sequence identity to SEQ ID NO: 1.

21. The cell of claim 20, wherein said variable heavy chain domain comprises the sequence of SEQ ID NO: 1.

22. The cell of any one of claims 16-21, wherein said third cell surface molecule further comprises an immunoglobulin light chain domain.

23. The cell of claim 22, wherein said immunoglobulin light chain domain has at least a 90% sequence identity to SEQ ID NO:2.

24. The cell of claim 22, wherein said immunoglobulin light chain domain comprises the sequence of SEQ ID NO:2.

25. The cell of claim 16, wherein said third cell surface molecule is a protein monomer, dimer, or multimer that comprises the amino acid sequences of both SEQ ID NO: 1 and SEQ ID NO:2.

26. The cell of any one of claims 16-25, wherein said third cell surface molecule has at least a 90% sequence identity to SEQ ID NO:7.

27. The cell of claim 26, wherein said third cell surface molecule has the sequence of SEQ ID NO:7.

28. The cell of claim 16, wherein said third cell surface molecule comprises an antibody Fab or a single chain variable fragment (scFV) that binds to CD200R.RUC027WSF2024-22329. The cell of claim 28, wherein said Fab or scFV binds to CD200R with an affinity measured by its dissociation constant (Kd), wherein said Kd is between about 107and 1013M.

30. The cell of any one of claims 16-29, wherein said third cell surface molecule is a fusion protein comprising a heterologous transmembrane domain (TMD).

31. The cell of claim 30, wherein said TMD comprises a single a helix, multiple a helices, or a rolled-up P sheet.

32. The cell of claim 30, wherein said TMD has at least a 90% sequence identity to SEQ ID NO:6.

33. The cell of claim 30, wherein said TMD has the sequence of SEQ ID NO:6.

34. The cell of claim 30, wherein said TMD is selected from the group consisting of CD99, CD47, CD64, CD85f, CD349, CD284, CD261, CD 172b, CD277, CD 186, CD 156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CDla, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221, CD271, CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CDl lb, CD107a, CD331, CD222, CD20, CD79a, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CDle, CDld, CD227, CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CDl la, CDl lc, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CDlb, CDlc, CD154, CD6, CD52, CD132, CD32, CD66b, CD171, CD191, CD197, CD185, CD131, CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361, CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD 110, CD79b, CD200, CD 192, CD231, CD86, CD212, CD 118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD158i, CD178, CD295, CD151, CD97, CD183, CD39, CD239, CD193, CD194, CD195, CD196,RUC027WSF2024-223CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241, CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91, CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161, CD215, CD150, CDl ld, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111, CD281, CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158z, CD158f2, CD85j, CD300f, CD92, CD351, CD112, CD 100, CD270, CD101, CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD 114, CD 180, CD158d, CD273, CD290, CD244, CD 169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201, CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51, CD41, CD29, CD 18, CD61, and CD 104.

35. The cell of claim 34, wherein said third cell surface molecule has at least a 90% sequence identity to SEQ ID NO:28.

36. The cell of claim 34, wherein said third cell surface molecule has the sequence of SEQ ID NO:28.

37. The cell of claim 16, wherein said third cell surface molecule does not have an intracellular domain (ICD) or does not transmit an intracellular signal.

38. The cell of any one of claims 16-22 wherein said third cell surface molecule further comprises a cytoplasmic domain that does not transmit an intracellular signal.

39. The cell of any one of claims 16-22, wherein said third cell surface molecule has an intracellular domain from CD16, CD32, CD64, CD8, CD3, CD28, CD137, SEQ ID NO:42, or SEQ ID NO:43.

40. The cell of claim 16, wherein said third cell surface molecule has CD200 activity and has at least a 90% sequence identity to SEQ ID NO:22.

41. The cell of claim 16, wherein said third cell surface molecule has the sequence of SEQ ID NO:22.

42. The cell of claim 16, wherein said third cell surface molecule has CD200 activity and comprises a CD200 extracellular domain with at least a 90% sequence identity to SEQ ID NO:29.

43. The cell of claim 16, wherein said third cell surface molecule has CD200 activity and comprises a CD200 extracellular domain having the sequence of SEQ ID NO:29.RUC027WSF2024-22344. The cell of any one of claims 1-43, further comprising a fourth cell-surface molecule that engages CD99 ligands.

45. The cell of claim 44, wherein said fourth cell-surface molecule has a CD99 extra-cellular function and has at least a 90% sequence identity to SEQ ID NO:21.

46. The cell of claim 45, wherein said fourth cell-surface molecule has the sequence of SEQ ID N0:21.

47. The cell of claim 44, wherein said fourth cell-surface molecule is a CD99 chimera comprising a CD99 extracellular domain and a heterologous transmembrane domain, wherein said fourth cell-surface molecule lacks CD99 intracellular signaling.

48. The cell of claim 47, wherein said heterologous transmembrane domain has at least a 90% sequence identity with SEQ ID NO:6.

49. The cell of claim 48, wherein said transmembrane domain has the sequence of SEQ ID NO:6.

50. The cell of claim 47, wherein said fourth cell-surface molecule has at least a 90% sequence identity with SEQ ID NO:30.

51. The cell of claim 50, wherein said fourth cell surface molecule has the sequence of SEQ ID NO:30.

52. The cell of any one of claims 1-51, wherein said cell has a reduced or eliminated cell surface leukocyte antigen class I.

53. The cell of claim 52, wherein said leukocyte antigen class I is the human leukocyte antigen class I (HLA-I).

54. The cell of claim 52, wherein said leukocyte antigen class I is the swine leukocyte antigen class I (SLA-I).

55. The cell of any one of claims 1-54, wherein said cell has a reduced or eliminated cell surface leukocyte antigen class II.

56. The cell of claim 55, wherein said leukocyte antigen class II is the human leukocyte antigen class II (HLA-II).

57. The cell of claim 55, wherein said leukocyte antigen class II is the swine leukocyte antigen class II (SLA-II).

58. The cell of any one of claims 1-57, wherein said cell is ABO blood group compatible with a subject into which said cell is transplanted.

59. The cell of claim 58, wherein said cell is ABO blood group type O.

60. The cell of any one of claims 1-59, wherein said cell is rhesus factor (Rh) blood group compatible with a subject into which said cell is transplanted.RUC027WSF2024-22361. The cell of claim 60, wherein said cell is Rh-.

62. The cell of any one of claims 1-61, wherein said cell is derived from a primary cell, a comprehensively immune protected (CIP) cell, a hypoimmune cell, a hypoimmune pluripotent (HIP) cell, a HIP cell that is ABO blood group O and Rhesus Factor negative (HIPO-), an induced pluripotent stem cell (iPSC), or an embryonic stem cell (ESC).

63. The cell of any one of claims 1-62, wherein said cell is selected from the group consisting of a chimeric antigen receptor (CAR) cell, an immune cell expressing an endogenous or exogenous T cell receptor (TCR), an immune cell, an endothelial cell, a dopaminergic neuron, a pancreatic islet cell, a cardiomyocyte, a retinal pigment endothelium cell, a thyroid cell, and a parathyroid cell.

64. The cell of any one of claims 1-63, wherein said cell is from a species selected from the group consisting of a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig.

65. The cell of claim 64, wherein said cell is from a human.

66. The cell of claim 65, wherein said cell is from a pig.

67. The cell of claim 66, wherein said pig cell comprises a knockout in one or more of a GGTA1, CMAH, B4GALNT2, or growth hormone receptor (GHR) gene.

68. The cell of any one of claims 64 to 67, wherein said cell comprises one or more of human Thrombomodulin (TBM), CD46, DAF, EPCR, HO-1, or CD59 transgenes.

69. The cell of claim 1, wherein said first cell surface molecule is a CD47 chimera, wherein said CD47 chimera comprises a CD47 extracellular domain bound to a cell surface of said cell by a heterologous transmembrane domain.

70. A method of treating a disease with the cell of any one of claims 1 to 69, comprising the step of transplanting said cell into a subject, wherein said cell evades killing by immune cells.

71. The method of claim 70, wherein said immune cells are leukocytes, myeloid cells, polymorphonuclear (PMN) cells, macrophages, T cells, cytotoxic T cells, or natural killer (NK) cells.

72. A method of treating a disease with the cell of any one of claims 1-69, comprising the step of transplanting said cell into a subject, wherein said cell is less susceptible to said subject’s immune rejection when compared to a parental cell.

73. The method of any one of claims 70-72, wherein said transplantation step utilizes a single-cell suspension of islet cells.I l lRUC027WSF2024-22374. The method of any one of claims 70-73, further comprising administering a danger- associated molecular pattern (DAMP) inhibitor, a DAMP pathway inhibitor, or a combination thereof.

75. The method of claim 74, wherein said DAMP inhibitor, DAMP pathway inhibitor, or said combination thereof are delivered to said subject using separate delivery methods.

76. The method of any one of claims 70-74, further comprising administering cyclosporin A (CsA), dexamethasone, an anti-SIRL-1 antibody, a soluble CD99 chimera, BAY 85-8501, alpha-1 antitrypsin (A1AT), TAK-242, apocynin, idelalisib, CpG-52364, colchicine, or the anti-TLR4 antibody NI-0101.

77. The method of any one of claims 70-76, further comprising administering the combination of an anti-SIRL-1 antibody and BAY 85-8501.

78. The method of any one of claims 70-76, further comprising administering the combination of CsA and BAY 85-8501.

79. A pharmaceutical composition, comprising the cell of any one of claims 1-69 and a pharmaceutical excipient.

80. The pharmaceutical composition of claim 80, further comprising Colchicine, Idelalisib, or oligonucleotide CpG-52364.

81. A medicament for treating a disease, comprising the cell of any one of claims 1-69 and a pharmaceutical excipient.

82. The medicament of claim 81, wherein said cell is selected from the group consisting of a chimeric antigen receptor (CAR) cell, an immune cell expressing an endogenous or exogenous T cell receptor (TCR), an immune cell, an endothelial cell, a dopaminergic neuron, a pancreatic islet cell, a cardiomyocyte, a retinal pigment endothelium cell, a thyroid cell, and a parathyroid cell.

83. The medicament of either one of claims 80-82, wherein said disease is selected from the group consisting of Type I Diabetes, Type II Diabetes, monogenic diabetes, a cardiac disease, a neurological disease, a cancer, an ocular disease, a vascular disease, and an endocrinologic disease.

84. A method of treating a disease with a tissue comprising the cell of any one of claims 1-69, comprising the step of transplanting said tissue into a subject, wherein said cell evades killing by said immune cells.

85. A method of treating a disease with an organ comprising the cell of any one of claims 1- 69, comprising the step of transplanting said organ into a subject, wherein said cell evades killing by said immune cells.RUC027WSF2024-22386. The method of any one of claims 70-78 or 84-85, wherein said cell and said subject are not of the same species.

87. The method of any one of claims 84-86, wherein said subject is a human.

88. The method of any one of claims 84-86, wherein said cell is from a species selected from the group consisting of a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig.

89. The method of claim 88, wherein said cell is a pig cell.

90. The method of claim 84, wherein said tissue and said subject are not of the same species.

91. The method of claim 90, wherein said subject is a human.

92. The method of claim 90, wherein said tissue is from a species selected from the group consisting of a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig.

93. The method of claim 90, wherein said tissue is a pig tissue.

94. The method of claim 85, wherein said organ and said subject are not of the same species.

95. The method of claim 94, wherein said subject is a human.

96. The method of claim 94, wherein said organ is from a species selected from the group consisting of a human, monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, and guinea pig.

97. The method of claim 96, wherein said organ is a pig organ.

98. A genetically-modified non-human animal, comprising a reduced or eliminated endogenous leukocyte antigen class I expression, wherein said genetically-modified non- human animal expresses a human CD200R engager (CD200RE), a human SIRPa engager (SIRPaE), a human Fc Sequestration molecule, and a human CD99 ligand engager (CD99LE).

99. The genetically -modified non-human animal of claim 98, further comprising a reduced or eliminated endogenous leukocyte antigen class II expression.

100. The genetically -modified non-human animal of either one of claims 98-99, wherein said genetically-modified non-human animal is a monkey, cow, pig, chicken, turkey, horse, sheep, goat, donkey, mule, duck, goose, buffalo, camel, yak, llama, alpaca, mouse, rat, dog, cat, hamster, or a guinea pig.RUC027WSF2024-223101. The genetically -modified non-human animal of any one of claims 98-100, wherein said genetically-modified non-human animal is a pig.

102. The genetically-modified non-human animal of any one of claims 98-101, wherein said animal comprises a knockout in one or more of a GGTA1, CMAH, B4GALNT2, or growth hormone receptor (GHR) gene.

103. The genetically -modified non-human animal of any one of claims 98-102, wherein said animal comprises human Thrombomodulin (TBM), CD46, DAF, EPCR, HO-1, or CD59 transgenes.

104. The genetically-modified non-human animal of any one of claims 98-103, wherein said genetically-modified non-human animal has been modified to eliminate the risk of transmitting porcine endogenous retroviruses (PERV).

105. The genetically -modified non-human animal of claim 104, wherein said risk has been eliminated by CRISPR.

106. The genetically -modified non-human animal of either one of any one of claims 104- 105, wherein said genetically-modified non-human animal is PERV-C negative.

107. A use of the cell of any one of claims 1 to 69 for transplanting into a subject for treating a disease, wherein said cell evades killing by immune cells.

108. A use of a tissue comprising the cell of any one of claims 1 to 69 for transplanting into a subject for treating a disease, wherein said cell evades killing by immune cells.

109. A use of an organ comprising the cell of any one of claims 1 to 69 for transplanting into a subject for treating a disease, wherein said cell evades killing by immune cells.

110. A use of the cell of any one of claims 1 to 69 for manufacturing a medicament for treating a disease, wherein said cell evades killing by immune cells.

111. A use of a tissue comprising the cell of any one of claims 1 to 69 for manufacturing a medicament for treating a disease, wherein said cell evades killing by immune cells.

112. A use of an organ comprising the cell of any one of claims 1 to 69 for manufacturing a medicament for treating a disease, wherein said cell evades killing by immune cells.

113. A method of killing the cell of any one of claims 1-69 in vivo, comprising administering a SIRPa antagonist to a subject into which said cell has been transplanted, wherein said cell expresses a cell-surface SIRPa engager.

114. The method of claim 113, wherein said SIRPa antagonist comprises an immunoglobulin domain.

115. The method of claim 113, wherein said SIRPa antagonist is an antibody or an scFv.

116. The method of claim 113, wherein said SIRPa antagonist is an antibody.RUC027WSF2024-223117. The method of any one of claims 113-116, wherein said antibody binds to said SIRPa with an affinity measured by its dissociation constant (Kd), wherein said Kd is between about 107and 1013M.

118. The method of claim 116, wherein said antibody has at least a 90% sequence identity to SEQ ID NO:46.

119. The method of claim 116, wherein said antibody has the sequence of SEQ ID NO:46.

120. The method of any one of claims 113-119, wherein said subject is a human.

121. The method of any one of claims 113-120, wherein said cell-surface SIRPa engager comprises CD47, a truncated CD47, a CD47 chimera, or an anti-SIRPa immunoglobulin domain.

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