Allograft tolerance without systemic immunosuppression

Abstract

Provided is a cell that is genetically modified to include at least one mechanism for providing local immunosuppression at the transplant site when transplanted into an allogeneic host; and methods for preparing and using the cell. The cell comprises a set of transgenes, each of which encodes a gene product that is cytoplasmic, membrane-bound or locally acting, and whose function is one or more of the following: reducing antigen presenting cell activation and function; reducing leukocyte activity or cytolytic function that attacks the graft; reducing macrophage cytolytic function and phagocytosis of allogeneic graft cells; inducing apoptosis in leukocytes that attack the graft; reducing local inflammatory proteins; and protecting against leukocyte-mediated apoptosis. The transgenes include two or more of the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or Fasl, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6.

Description

Field of the Invention

[0001] The present disclosure relates generally to the field of transplantation. The present disclosure further relates to methods for generating local immunosuppression in the environment of transplanted cells. Background of the Invention

[0003] The advent of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells has had a paradigm shifting effect on regenerative and translational medicine. These cells can self-renew indefinitely in a pluripotent state while retaining the ability to differentiate into any cell type in the human body. Such properties allow researchers to better understand the causes of human development and developmental disorders. They also provide modern medicine with powerful new tools for diseases that are difficult or impossible to treat with conventional medicine, including spinal cord injury, diabetes, blindness, multiple sclerosis, and cancer, to name a few. As the understanding of how to control stem cell differentiation and the biology of differentiated cell products continues to increase, the efficacy and range of applicable diseases of cell therapies will only increase.

[0004] These applications bring important and severe challenges. Together with cell safety, one of the most important concerns is the immune rejection of cells from different genetic backgrounds. Immune rejection remains a key obstacle because the immune system has evolved a set of complex mechanisms to identify and eliminate "non-self" cells expressing specific protein fragments-especially those from the major histocompatibility complex (MHC in mice, HLA in people)-different between donors and recipients (Yang et al., Nat Rev Genet.18:309-26 (2017)). This response is almost certainly a byproduct of the evolutionary pressure to protect against opportunistic infections and malignancies, which are generally defined by the presence of "foreign" proteins and epitopes. Depending on the circumstances, the rejection of transplanted cells or tissues can occur in a time scale of minutes / hours (hyperacute), days / months (acute) and months / years (chronic) (LaRosa et al., J Immunol.178:7503-9 (2007)). This rejection results from complex and coordinated effects of cell types from both innate (Murphy et al., Immunol Rev. 241:39-48 (2011)) and adaptive immunity (Issa et al., Expert Rev Clin Immunol. 6:155-69 (2010)).

[0005] One of the most important pathways of rejection is the initiation of the adaptive immune system and the activation of CD8+ cytotoxic T cells. This occurs after antigen presenting cells process donor-specific peptides and then activate recipient T cells that are specific for the same peptides in secondary lymphoid organs (Lechler et al., J Exp Med. 155: 31-41 (1982); Guermonprez et al., Annu Rev Immunol. 20: 621-67 (2002); Stockwin et al., Immunol Cell Biol. 78: 91-102 (2000)). These T cells then migrate to and kill transplanted cells or tissues, accompanied by the release of cytolytic factors such as perforin and granzymes. NK cells can also induce apoptosis of donor cells based on foreign MHC expression or absence of MHC expression (Kitchens et al., Transplantation. 81:811-7 (2006); Benichou et al., Curr Opin Organ Transplant. 16:47-53 (2011)), and other cell types such as macrophages can support rejection by releasing proinflammatory cytokines at the implant site (Mannon, Curr Opin Organ Transplant. 17:20-5 (2012)). Many other cell types and subtypes also play a role in allograft rejection. Since these are the same immune pathways used to eliminate common viral and bacterial pathogens, they - along with allograft rejection - are highly conserved across vertebrate species.

[0006] Current solutions for preventing allograft rejection involve two options: finding a donor with a matching histocompatibility haplotype (most likely from a genetically related family) and, much more commonly, using broadly targeted immunosuppressant drugs (Wiseman, Clin J Am Soc Nephrol. 11:332-43 (2016); Malaise et al., Transplant Proc. 37:2840-2 (2005)). Common drugs include those from the following families: calcineurin inhibitors (Flechner et al., Clin Transplant. 22: 1-15 (2008); Casey et al., Curr Opin Nephrol Hypertens. 20: 610-5 (2011)), antiproliferative agents (Hardinger et al., World J Transplant. 3: 68-77 (2013)), mTOR inhibitors (Macdonald, Expert Rev Clin Immunol. 3: 423-36 (2007); Neuhaus et al., Liver Transpl. 7: 473-84 (2001)) and steroids (Steiner et al., Semin Immunopathol. 33: 157-67 (2011)) - all of which suppress T cell proliferation or function (especially the first three). These drugs need to be taken daily for life, and even missing a single dose can increase the risk of rejection. However, they do not always work, and when they do, chronic rejection rates continue to rise over time (Demetris et al., Ann Transplant. 2:27-44 (1997); Libby et al., Immunity. 14:387-97 (2001)). Most importantly, they have systemic effects and ultimately leave patients immunocompromised, with increased rates of cancer and life-threatening infections (Gallagher et al., J Am Soc Nephrol. 21:852-8 (2010)). With respect to ES cells, these drugs have shown only minor improvements in allowing survival across the MHC barrier (Swijnenburg et al., Proc Natl Acad Sci USA. 105:12991-6 (2008); Toriumi et al., Neurol Res. 31:220-7 (2009)).Although newer and more targeted immunosuppressant agents are becoming available and being tested in the skin and heart (Larsen et al., Nature. 381:434-8 (1996)), as well as in ES cell allograft settings (Pearl et al., Cell Stem Cell. 8:309-17 (2011), they still exert systemic effects and therefore may compromise the host's immunity.

[0007] A proposed benefit of the discovery of iPS cells is that they can be produced from each patient and for each patient. In theory, these cells should be protected from the immune rejection of the corresponding patients (Pearl et al., Sci Transl Med.4:164ps25 (2012)). However, inducing the iPS cell state involves epigenetic changes and in vitro culture pressure that can produce abnormalities and malignancies, so each cell line needs to be strictly tested and / or genetically modified to achieve safety and function (Hussein et al., Nature.471:58-62 (2011); Laurent et al., Cell Stem Cell.8:106-18 (2011); Lister et al., Nature.471:68-73 (2011)). Ultimately, the cost and time required to produce and test iPS cell lines for each individual patient make this method practically and economically impractical. Even if the cost is greatly reduced, it is impossible to help those patients who need to immediately treat diseases such as burns, heart attacks, strokes and spinal cord injuries (etc.). Furthermore, in light of recent findings, whether iPS cell-derived cell types are truly protected from immune rejection even when transplanted into the same host from which they were derived remains controversial (Zhao et al., Nature. 474:212-5 (2011)).

[0008] In this regard, a proposed solution is to use natural suppressive or regulatory immune cells, such as Treg or others, which are expanded and / or transferred before, during or after the transplantation of therapeutic cells or tissues (Cobbold et al., Cold Spring Harb Perspect Med.3(6)(2013); Wood et al., Nature reviews Immunology.12:417-30(2012)). These strategies have been proposed based on the recognition of suppressive immune pathways, especially the discovery of the master regulator FoxP3 that programs a subset of CD4+ cell regulatory T cells (Hori et al., 299:1057-61(2003); Fontenot et al., Nat Immunol.4:330-6(2003)) and their evidence of vital importance in promoting tolerance to allogeneic transplants (Kendal et al., J Exp Med.208:2043-53(2011)). This idea contrasts with some of the initial tolerance induction strategies, which were almost entirely focused on depleting effector T cells with monoclonal antibodies, combined with bone marrow transplants and the generation of donor chimerism (Cobbold et al., Nature. 323: 164-6 (1986); Qin et al., J Exp Med. 169: 779-94 (1989)). The importance of the suppressive T cell phenotype was later realized, along with strategies that would not kill the cells, but block key T cell receptors in a way that would render them unresponsive to allogeneic transplants (Cobbold et al., J Immunol. 172: 6003-10 (2004)), but at the same time be able to suppress naive T cells with other specificities (Cobbold et al., Immunol Rev. 129: 165-201 (1992); Qin et al., Eur J Immunol. 20: 2737-45 (1990)).These cells, now identified as Tregs, can promote tolerance through a variety of mechanisms, including, but not limited to, suppressive factors such as TGFβ (Nakamura et al., The Journal of experimental medicine. 194:629-44 (2001); Nakamura et al., J Immunol. 172:834-42 (2004)), CTLA4 (Tang et al., J Immunol. 181:1806-13 (2008); Walker et al., Trends Immunol. 36:63-70 (2015)), IL10 (O'Garra et al., J Clin Invest. 114:1372-8 (2004); Chaudhry et al., Immunity. 34:566-78 (2011)) and IL35 (Collison et al., Nature. 450:566-9 (2007)), as well as preferential depletion of IL-2 (Shevach et al., Immunity. 30:636-45 (2009); Setoguchi et al., J Exp Med. 201:723-35 (2005)), manipulation or killing of antigen presenting cells (Mahnke et al., Cell Immunol. 250:1-13 (2007); Shevach et al., Immunol Rev. 212:60-73 (2006)) and local ATP (Regateiro et al., Eur J Immunol. 41:2955-65 (2011); Regateiro et al., Clin Exp Med. 201:723-35 (2005)). Immunol. 171: 1-7 (2013)) or depletion of essential amino acids (Cobbold et al., Proc Natl Acad Sci USA. 106: 12055-60 (2009)).

[0009] Two methods of the potential therapeutic use of Treg involve the use of in vitro expansion of donor antigens together with transplantation, or the selective in vivo expansion using the difference between regulatory T cells and effector T cells. Although these strategies are interesting, so far, only by using Treg expanded in vitro or in vivo can not prove long-term acceptance of allogeneic transplants. Treg biology still has many complications and unknown aspects, including the best method for in vitro or in vivo expansion, and the dosage and timing of treatment-related. It has also been shown that, depending on the inflammatory background, antigen-specific Treg can be "defeated" to contain (Korn et al., Nat Med. 13: 423-31 (2007)), and Treg can be killed by NK cells (Roy et al., J Immunol. 180: 1729-36 (2008)).

[0010] In addition to Treg, other suppressive cell types have been explored to induce allograft tolerance, such as antigen presenting cells, like dendritic cells (DC) (Walker et al., Trends Immunol. 36: 63-70 (2015)). DC is the link between innate immunity and adaptive immunity, and they can induce effector and suppressive immune responses depending on the situation such as their maturation state and local inflammation prompts. During allograft rejection, DC presents allograft antigens to the binding groove of MHC (mouse) or HLA (human) molecules on its surface together with costimulatory molecules such as CD80, CD86 and CD40 (etc.), and allograft-specific T cell clones recognize the molecules and become activated (Walker et al., Trends Immunol. 36: 63-70 (2015)). Tolerogenic DCs can be induced from an immature state by exposure to suppressive cues that keep expression levels of MHC and co-stimulatory molecules low and, in turn, promote naive T cells to anergic or even T regulatory subtypes following DC-T cell interactions.

[0011] Therapeutically, one application of this biology is to expand DCs that are simultaneously exposed in vitro to specific target allograft antigens and immunosuppressive factors (many of which have been tested, including TGF-β, IL10, cAMP, prostaglandin E2, histamine, neuropeptides, vitamin D2, B2 agonists, HLA-G, glucosamine) as well as drugs such as corticosteroids, cyclosporine, tacrolimus, rapamycin, aspirin, mycophenolate mofetil, sanglifehrin, and deoxyspergualin (Hackstein et al., Nat Rev Immunol. 4:24-34 (2004)). Alternatively, DCs have been genetically engineered to directly express immunomodulatory factors such as TGF-β, IL-10, VEGF, FasL, CTLA4-Ig, IDO, NFKb decoy receptors, soluble TNFR, CCR7, and siRNA-induced IL-silencing 12 (Morelli et al., Immunol Rev. 196: 125-46 (2003)). These cultured or engineered DCs are then transferred into recipients along with allografts to test whether they can prolong allograft survival, assuming that they suppress allograft-specific T cells or increase the number of T regulatory cells concentrated in the allograft.

[0012] In one prototype approach of this type, bone marrow-derived DCs were transduced with SOCS1 (thus preventing upregulation of co-stimulatory molecules and MHCII), which extended mouse cardiac allografts (Fu et al., Cell Mol Immunol. 6:87-95 (2009)). In another demonstration, DCs expressing FasL were also able to extend mouse cardiac allografts (Min et al., J Immunol. 164:161-7 (2000)). In general, there are many single and combined approaches using tolerogenic DCs following these approaches (Bjorck et al., J Heart Lung Transplant. 24: 1118-20 (2005); Sun et al., PLoS One. 7: e52096 (2012); Li et al., J Immunol. 178: 5480-7 (2007); Xu et al., Transplant Proc. 38: 1561-3 (2006); Lan et al., J Immunol. 177: 5868-77 (2006); Lutz et al., Eur J Immunol. 30: 1813-22 (2000); Fischer et al., Transpl Immunol. 25: 20-6 (2011)), and the results are highly variable depending on the type of modification of the DCs, culture conditions, timing, and type of allograft tested (Zhou et al., J Immunol. 38: 1561-3 (2006); Lan et al., J Immunol. 177: 5868-77 (2006); Lutz et al., Eur J Immunol. 30: 1813-22 (2000); Fischer et al., Transpl Immunol. 25: 20-6 (2011)). Res. 2016: 5730674 (2016); Xia et al., J Evid Based Med. 7: 135-46 (2014)). Almost all of these studies have been conducted in mice, but recent human testing has begun, including testing for safety in healthy volunteers (Dhodapkar et al., J Exp Med. 193: 233-8 (2001); Dhodapkar et al., Blood. 100: 174-7 (2002)) and a Phase I clinical trial in 10 diabetic patients (Giannoukakis et al., Diabetes Care. 34: 2026-32 (2011)).

[0013] There are still many unknowns for adoptive Treg and tolerogenic DC therapies, and the most important one is the duration of their efficacy. Although in vivo studies have shown that extended allograft survival is possible using these two methods (with or without additional immunosuppressive drugs), it is not long-term, and in almost every case, allografts eventually die. This is consistent with the fact that both Treg and DC have a limited time span. In addition, especially in DC, the tolerogenic phenotype is likely to "convert" and promote inflammatory pathways (Delamarre et al., SeminImmunol.23:2-11 (2011); Schreibelt et al., Cancer Immunol Immunother.59:1573-82 (2010); Satpathy et al., Nat Immunol.14:937-48 (2013)). This may be due to the highly adaptive nature of DC and their ability to sense and respond to various inflammatory cues. It is also shown that these cells can die very quickly after adoptive transfer in vivo. There are also many subsets of suppressive Tregs and tolerogenic DCs that have been described, and it is not yet clear which is the ideal subset, or whether it will depend entirely on the context of allograft transplantation.

[0014] In addition, these kinds of methods present huge practical and economic barriers because they require clinicians to manipulate and handle complex immune cell types in addition to therapeutic methods. Given their limited lifespan, it is still unclear whether continuous and / or repeated delivery of these cells is required to confer long-term tolerance to allografts. This will complicate the already expensive and timely methods for culturing, amplifying or transducing cells with key immunomodulatory factors and ultimately hinder the use of extremely time-sensitive treatments.

[0015] Another approach for inducing tolerance is the use of hematopoietic cell transplantation (HCT), in which recipients of HLA-mismatched organs receive HCT using hematopoietic cells from the same donor (Gozzo et al., Surg Forum. 21:281-4 (1970); Ildstad et al., Nature. 307:168-70 (1984); Sayegh et al., Ann Intern Med. 114:954-5 (1991); Huang et al., J Clin Invest. 105:173-81 (2000); Kawai et al., N Engl J Med. 358:353-61 (2008); Sachs et al., Semin Immunol. 23:165-73 (2011)). This creates chimerism that allows newly developed T and B cells in the recipient to tolerate both recipient and donor antigens (Tomita et al., J Immunol. 153: 1087-98 (1994); Tomita et al., Transplantation. 61: 469-77 (1996); Tomita et al., Transplantation. 61: 477-85 (1996); Khan et al., Transplantation. 62: 380-7 (1996); Manilay et al., Transplantation. 66: 96-102 (1998)). This is due to the role played by hematopoietic cells in positive and negative selection in the thymus, where they eliminate cells with affinity for antigens containing hematopoietic cells that may also be present in the allograft, ultimately leading to their rejection. (Griesemer et al., Transplantation. 90: 465-74 (2010)). However, the inherent and dangerous risk of this method is the possibility of graft-versus-host disease (GVHD), in which the transplanted hematopoietic cells recognize the recipient tissue as foreign and carry out systemic attack (Sun et al., PLoS One.7:e52096 (2012)). From the beginning, several HCT variants have been developed to suppress rejection, including the use of non-myeloablative strategies. These strategies use modified chemotherapy regimens, generally involving lower doses, so that the recipients receiving HCT will not receive complete elimination of their hematopoietic compartments. The latest strategies in these strategies, for example, use HCT based on promoting tolerance cells (FC) to promote tolerance in HLA-mismatched kidney recipients, while largely avoiding GHVD (Leventhal et al., Sci Transl Med.4:124ra28 (2012)).

[0016] In addition to the risk of GHVD and the risk of secondary HCT procedures, the general limitation of these methods of inducing chimerism is that HLA-matched donors are required to be available for collecting bone marrow. Although this is easily achieved in rodent studies, it is a rather harsh requirement in people. Ideally, donor organs should be obtained from donors as soon as possible, which will leave a very short window (if possible) for collecting bone marrow from them. This is also an expensive and logistically demanding procedure that requires extremely patient- and operation-specific methods. And, like regulatory cell methods, it is unclear how to actually apply it to those situations where patients can benefit from or need to immediately treat the therapy of acute injuries or diseases.

[0017] Another approach that has been tested for reducing allograft rejection in vivo is to remove tissue compatibility molecules (Torikai et al. Blood. 122: 1341-9 (2013)), which are the main source of antigens recognized by "non-self" in allograft rejection. This is consistent with empirical data that HLA-matched donors and recipients greatly improve organ survival after transplantation (Opelz et al., Rev Immunogenet. 1: 334-42 (1999)). Although some positive results have been achieved using this approach, removing class I MHC makes cells extremely susceptible to NK cells (Pegram et al., Immunol Cell Biol. 89: 216-24 (2011); Raulet et al., Nat Rev Immunol. 6: 520-31 (2006); Huntington, Immunol Cell Biol. 92: 208-9 (2014)). It also leaves intact the MHC-independent killing pathway among CD8+ T cells (Haspot et al., Am J Transplant. 14:49-58 (2014)) and does not resolve antigenic differences outside the MHC / HLA gene family (minor antigens) (Roopenian et al., Immunol Rev. 190:86-94 (2002)).

[0018] The adoption of this method involves the deletion of all classical HLA class I molecules from pluripotent stem cells, combined with the introduction of genes encoding HLA-E (a minimal polymorphic HLA that inhibits NK cells) (Gornalusse et al., Nat Biotechnol. (2017)). Although this method shows short-term resistance to NK and CD8 T cell attacks in partially immunocompromised humanized mice, it has not been shown that these cells can survive for a long time in a host with full immune competence. In another method, ES cells are engineered to express PD-L1 and CTLA4-Ig, which improves survival in allogeneic hosts (Rong et al., Cell Stem Cell. 14: 121-30 (2014)), but with serious limitations, i.e. CTLA4-Ig can lead to systemic immune suppression. It has not yet been demonstrated that a set of modifications to ES or iPS cells allows them to escape allogeneic transplant rejection without the possibility of systemic immune suppression and without the need for immunosuppressive drugs.

[0019] It is an object of the present disclosure to mitigate and / or eliminate one or more of the above mentioned disadvantages. Summary of the invention

[0020] In one aspect, a cell is provided, which is genetically modified to contain at least one mechanism for providing local immune suppression at the transplant site when transplanted into an allogeneic host. The genetically modified cell comprises: a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane-bound or locally acting; and has one or more of the following functions: a) reducing antigen presenting cell activation and function; b) reducing leukocyte activity or cytolytic function that attacks the transplant; c) reducing macrophage cytolytic function and phagocytosis of allogeneic transplant cells; d) inducing apoptosis of leukocytes that attack the transplant; e) reducing local inflammatory proteins; and f) protecting against leukocyte-mediated apoptosis.

[0021] In one embodiment of the cell, the set of transgenes includes one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the following genes: PD-L1, HLA-G (or the mouse form of HLA-G, H2-M3), Cd47, Cd200, FASLG (or the mouse form of FASLG, FasL), Ccl21 (or the mouse form of Ccl21, Ccl21b), Mfge8, and Serpin B9 (or the mouse form of Serpin B9, Spi6).

[0022] In one embodiment of the cell, the set of transgenes includes two or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0023] In one embodiment of the cell, the set of transgenic genes includes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6), or genes encoding biologics that act as agonists of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6).

[0024] In one embodiment of the cell, the cell further comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or all eleven) of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or a gene encoding a biologic that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.

[0025] In one embodiment of the cell, the TGF-β or biologic acts locally in the transplant environment. In one embodiment of the cell, the TGF-β or biologic acts locally in the transplant environment with minimal systemic effects.

[0026] In various embodiments of the cell, the cell is a stem cell, a cell suitable for genome editing, and / or a source of therapeutic cell types (e.g., a cell that can be differentiated into a therapeutic cell type or a cell of a desired target tissue). In various embodiments, the cell is an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem cell or progenitor cell, a germline stem cell, a lung stem cell or progenitor cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, an intestinal stem cell or progenitor cell, a pluripotent stem cell, an amniotic membrane stem cell, an umbilical cord blood stem cell, a neural stem cell or progenitor cell, an adult stem cell, a somatic stem cell, a tissue-specific stem cell, a totipotent stem cell, a fibroblast, a monocyte precursor, a B cell, an exocrine cell, a pancreatic progenitor cell, an endocrine progenitor cell, a hepatoblast, a myoblast, a preadipocyte, a hepatocyte, a chondrocyte, a smooth muscle cell, a K562 human erythroleukemia cell line, a bone cell, a synovial cell, a tendon cell, a ligament cell, a meniscus cell, a fat cell, Dendritic cells, natural killer cells, skeletal muscle cells, cardiomyocytes, erythrocytes-megakaryocytes, eosinophils, macrophages, T cells, pancreatic beta cells, neurons, cardiomyocytes, blood cells, exocrine progenitor cells, duct cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, bile duct cells, white or brown fat cells, hormone secreting cells, epidermal keratinocytes, epithelial cells, kidney cells, skeletal joint synovial cells, periosteal cells, perichondrial cells, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amniotic or placental membranes, serosal cells, somatic cells, or cells derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach.

[0027] In one embodiment of the cell, the cell is further genetically modified to include at least one (e.g., one, two, three or more) mechanism for controlling cell proliferation (e.g., to reduce the tumorigenic potential of the modified cell or to reduce the proliferation of the modified cell that has become tumorigenic). The genetically modified cell comprises: genetic modification of one or more (e.g., one, two, three or more) cell division loci / locus (CDL), wherein the CDL is one or more loci whose transcription products are expressed by dividing cells (e.g., all dividing cells contain one or more of the immunosuppressive transgenes), wherein the genetic modification is one or more of the following: a) an Eliminate Link (ALINK) system comprising a DNA sequence encoding a negative selectable marker transcriptionally linked to a DNA sequence encoding the CDL; and b) an Exogenous Activator (EARC) system regulated by a CDL, wherein the EARC system comprises a gene expression system based on an inducible activator operably linked to the CDL.

[0028] In one embodiment of the cell, the genetic modification of the CDL comprises targeted replacement of the CDL with one or more of the following: a) a DNA vector comprising the ALINK system; b) a DNA vector comprising the EARC system; c) a DNA vector comprising the ALINK system and the EARC system; wherein the ALINK and / or EARC system are each operably linked to the CDL.

[0029] In various embodiments of the cell, the ALINK genetic modification of the CDL is homozygous, heterozygous, hemizygous or compound heterozygous, and / or the EARC genetic modification ensures that functional CDL modifications can only be produced by EARC modified alleles.

[0030] In various embodiments of the cell, the CDL is one or more (e.g., one, two, three or more) of the loci listed in Table 5. In various embodiments, the CDL encodes a gene product that functions in one or more of the following: cell cycle, DNA replication, RNA transcription, protein translation, and metabolism. In various embodiments, the CDL is one or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5, and Eef2 / EEF2, preferably the CDL is Cdk1 or CDK1. In some embodiments, the CDL is Top2A. In some embodiments, the CDL is Eef2. In various embodiments, the CDL is two or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5, and Eef2 / EEF2, preferably the CDL is Cdk1 and Top2A or Cdk1 and Eef2.

[0031] In various embodiments of the cell, the ALINK system comprises a herpes simplex virus-thymidine kinase / ganciclovir system, a cytosine deaminase / 5-fluorocytosine system, a carboxylesterase / irinotecan system or an iCasp9 / AP1903 system, preferably the ALINK system is a herpes simplex virus-thymidine kinase / ganciclovir system.

[0032] In various embodiments of the cell, the EARC system is a doxycycline-inducible "dox-bridge" system, a coumarate switch-inducible system, an ecdysone-inducible system, a radio wave-inducible system or a ligand-reversible dimerization system, preferably the EARC system is a dox-bridge system.

[0033] In one aspect, a method for providing local immunosuppression at a transplant site in an allogeneic host is provided. The method comprises providing cells; and expressing a set of transgenes in the cells, each transgene encoding a gene product that acts cytoplasmically, membrane-bound or locally; and having one or more of the following functions: a) reducing antigen presenting cell activation and function; b) reducing leukocyte activity or cytolytic function that attacks the transplant; c) reducing macrophage cytolytic function and phagocytosis of allogeneic transplant cells; d) inducing apoptosis of leukocytes that attack the transplant; e) reducing local inflammatory proteins; and f) protecting against leukocyte-mediated apoptosis.

[0034] In one embodiment of the method, the set of transgenes includes one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6), or a gene encoding a biologic that acts as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, or Serpin B9 (Spi6).

[0035] In one embodiment of the method, the set of transgenes includes two or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and SerpinB9 (Spi6).

[0036] In one embodiment of the method, the set of transgenic genes includes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6), or genes encoding biologics that act as agonists of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and SerpinB9 (Spi6).

[0037] In one embodiment of the method, the method further comprises expressing one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or all eleven) of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or encoding a gene of a biological agent that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39. In one embodiment, the TGF-β or biological agent acts locally in the transplant environment. In one embodiment of the cell, the TGF-β or biological agent acts locally in the transplant environment with minimal systemic effect.

[0038] In various embodiments of the method, the cell is a stem cell, a cell suitable for genome editing, and / or a source of therapeutic cell types (e.g., a cell that can be differentiated into a therapeutic cell type or a cell of a desired target tissue). In various embodiments, the cell is an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem cell or progenitor cell, a germline stem cell, a lung stem cell or progenitor cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, an intestinal stem cell or progenitor cell, a pluripotent stem cell, an amniotic membrane stem cell, an umbilical cord blood stem cell, a neural stem cell or progenitor cell, an adult stem cell, a somatic stem cell, a tissue-specific stem cell, a totipotent stem cell, a fibroblast, a monocyte precursor, a B cell, an exocrine cell, a pancreatic progenitor cell, an endocrine progenitor cell, a hepatoblast, a myoblast, a preadipocyte, a hepatocyte, a chondrocyte, a smooth muscle cell, a K562 human erythroleukemia cell line, a bone cell, a synovial cell, a tendon cell, a ligament cell, a meniscus cell, a fat cell, Dendritic cells, natural killer cells, skeletal muscle cells, cardiomyocytes, erythrocytes-megakaryocytes, eosinophils, macrophages, T cells, pancreatic beta cells, neurons, cardiomyocytes, blood cells, exocrine progenitor cells, duct cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, bile duct cells, white or brown fat cells, hormone secreting cells, epidermal keratinocytes, epithelial cells, kidney cells, skeletal joint synovial cells, periosteal cells, perichondrial cells, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amniotic or placental membranes, serosal cells, somatic cells, or cells derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach.

[0039] In various embodiments of the method, the cells are provided (e.g., injected) to or near the site of transplantation. In various embodiments of the method, the cells are provided (e.g., injected or implanted) into a transplant (e.g., injected or implanted into a tissue or organ transplant before, during, or after transplantation). In some embodiments, the cells expressing the transgene are cells of the transplant (e.g., cells of the transplanted tissue or organ are modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0040] In one aspect, a method for controlling cell proliferation at a transplant site in an allogeneic host is provided (e.g., reducing the tumorigenic potential of cells at the transplant site or reducing the proliferation of cells that have become tumorigenic at the transplant site). The method comprises: a) genetically modifying one or more (e.g., one, two, three or more) cell division loci / locus (CDL) in the cell, wherein the CDL is one or more loci whose transcription products are expressed by dividing cells (e.g., all dividing cells contain one or more of the immunosuppressive transgenes), the genetic modification of the CDL comprising one or more of the following: i) an Elimination Link (ALINK) system comprising a DNA sequence encoding a negative selectable marker transcriptionally linked to a DNA sequence encoding the CDL; and i) a CDL-regulated inducible exogenous activator (EARC) system comprising an inducible activator-based gene expression system operably linked to the CDL; b) genetically modifying the cell to comprise at least one mechanism for providing local immunosuppression at the transplant site; c) genetically modifying the cell or the CDL to comprise at least one mechanism for providing local immunosuppression at the transplant site; said cell population being transplanted at a transplantation site in an allogeneic host; and d) allowing the proliferation of genetically modified cells comprising the ALINK system by maintaining said genetically modified cells comprising the ALINK system in the absence of an inducer of said negative selective marker, and / or eliminating and / or inhibiting the proliferation of cells comprising the ALINK system by exposing said genetically modified cells comprising the ALINK system to an inducer of said inducible activating factor-based gene expression system; and / or allowing the proliferation of genetically modified cells comprising the EARC system by exposing said genetically modified cells comprising the EARC system to an inducer of said inducible activating factor-based gene expression system, or preventing or inhibiting the proliferation of cells comprising the EARC system by maintaining said genetically modified cells comprising the EARC system in the absence of an inducer of said inducible activating factor-based gene expression system.

[0041] In one embodiment of the method, the genetic modification of the CDL comprises targeted replacement of the CDL with one or more of the following: a) a DNA vector comprising the ALINK system; b) a DNA vector comprising the EARC system; c) a DNA vector comprising the ALINK system and the EARC system; wherein the ALINK and / or EARC system are each operably linked to the CDL.

[0042] In various embodiments of the method, the ALINK genetic modification of the CDL is homozygous, heterozygous, hemizygous or compound heterozygous, and / or the EARC genetic modification ensures that functional CDL modifications can only be produced by ERAC modified alleles.

[0043] In various embodiments of the method, the CDL is one or more (e.g., one, two, three or more) of the loci listed in Table 5. In various embodiments, the CDL encodes a gene product whose function is related to one or more of the following: cell cycle, DNA replication, RNA transcription, protein translation and metabolism. In various embodiments, the CDL is one or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5 and Eef2 / EEF2, preferably the CDL is Cdk1 or CDK1. In some embodiments, the CDL is Top2A. In some embodiments, the CDL is Eef2. In various embodiments, the CDL is two or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5 and Eef2 / EEF2, preferably the CDL is Cdk1 and Top2A or Cdk1 and Eef2.

[0044] In various embodiments of the method, the ALINK system comprises a herpes simplex virus-thymidine kinase / ganciclovir system, a cytosine deaminase / 5-fluorocytosine system, a carboxylesterase / irinotecan system or an iCasp9 / AP1903 system, preferably the ALINK system is a herpes simplex virus-thymidine kinase / ganciclovir system.

[0045] In various embodiments of the method, the EARC system is a doxycycline-inducible "dox-bridge" system, a coumarate switch-inducible system, an ecdysone-inducible system, a radio wave-inducible system, or a ligand-reversible dimerization system, preferably the EARC system is a dox-bridge system.

[0046] In one embodiment of the method, the genetically modified cells comprise: a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane-bound or locally acting; and having one or more of the following functions: a) reducing antigen presenting cell activation and function; b) reducing leukocyte activity or cytolytic function that attacks the transplant; c) reducing macrophage cytolytic function and phagocytosis of allogeneic transplant cells; d) inducing apoptosis of leukocytes that attack the transplant; e) reducing local inflammatory proteins; and f) protecting against leukocyte-mediated apoptosis.

[0047] In one embodiment of the method, the set of transgenes includes one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6), or a gene encoding a biologic that acts as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, or Serpin B9 (Spi6).

[0048] In one embodiment of the method, the set of transgenes includes two or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and SerpinB9 (Spi6).

[0049] In one embodiment of the method, the set of transgenic genes includes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6), or genes encoding biologics that act as agonists of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and SerpinB9 (Spi6).

[0050] In one embodiment of the method, the cell further comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or all eleven) of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or a gene encoding a biologic that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.

[0051] In one embodiment of the method, the TGF-β or biologic acts locally in the transplant environment. In one embodiment, the TGF-β or biologic acts locally in the transplant environment with minimal systemic effects.

[0052] In various embodiments of the method, the cell is a stem cell, a cell suitable for genome editing, and / or a source of therapeutic cell types (e.g., a cell that can be differentiated into a therapeutic cell type or a cell of a desired target tissue). In various embodiments, the cell is an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem cell or progenitor cell, a germline stem cell, a lung stem cell or progenitor cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, an intestinal stem cell or progenitor cell, a pluripotent stem cell, an amniotic membrane stem cell, an umbilical cord blood stem cell, a neural stem cell or progenitor cell, an adult stem cell, a somatic stem cell, a tissue-specific stem cell, a totipotent stem cell, a fibroblast, a monocyte precursor, a B cell, an exocrine cell, a pancreatic progenitor cell, an endocrine progenitor cell, a hepatoblast, a myoblast, a preadipocyte, a hepatocyte, a chondrocyte, a smooth muscle cell, a K562 human erythroleukemia cell line, a bone cell, a synovial cell, a tendon cell, a ligament cell, a meniscus cell, a fat cell, Dendritic cells, natural killer cells, skeletal muscle cells, cardiomyocytes, erythrocytes-megakaryocytes, eosinophils, macrophages, T cells, pancreatic beta cells, neurons, cardiomyocytes, blood cells, exocrine progenitor cells, duct cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, bile duct cells, white or brown fat cells, hormone secreting cells, epidermal keratinocytes, epithelial cells, kidney cells, skeletal joint synovial cells, periosteal cells, perichondrial cells, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amniotic or placental membranes, serosal cells, somatic cells, or cells derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach.

[0053] In various embodiments of the method, the cells are provided (e.g., injected) to or near the site of transplantation. In various embodiments of the method, the cells are provided (e.g., injected or implanted) into a transplant (e.g., injected or implanted into a tissue or organ transplant before, during, or after transplantation). In some embodiments, the cells expressing the transgene are cells of the transplant (e.g., cells of the transplanted tissue or organ are modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0054] In various embodiments of the methods, the allogeneic host is a mammal. In various embodiments of the methods, the allogeneic host is a mouse or a human.

[0055] In various embodiments of the method, the host has a degenerative disease or disorder treatable with cell therapy. In various embodiments, the disease or disorder is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, nervous system disease (such as Parkinson's disease, Alzheimer's disease, Huntington's disease and ALS), enzyme or hormone deficiency, metabolic disorder (e.g., lysosomal storage disease, galactosemia, maple syrup urine disease, phenylketonuria, glycogen storage disease, mitochondrial disorder, Friedreich's ataxia, Peroxisomal disorders, disorders of metal metabolism or organic acidemias, autoimmune diseases (e.g., psoriasis, systemic lupus erythematosus, Graves' disease, inflammatory bowel disease, Addison's disease, Sjogren's syndrome), Hashimoto's thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infectious diseases, hemophilia, degenerative diseases (e.g., Charcot-Marie-Tooth disease, disease, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, cystic fibrosis, cytochrome C oxidase deficiency, Ehlers-Danlos syndrome, essential tremor, fibrodysplasia ossificans progressiva, infantile neuroaxonal dystrophy, keratoconus, keratoglobose, muscular dystrophy, neuronal ceroid lipofuscinosis, preexisting disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age-related disease (e.g., atherosclerosis, cardiovascular disease (e.g., angina, myocardial infarction), cataracts, osteoporosis, or hypertension).

[0056] In some embodiments of any of the foregoing aspects, one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level equal to or greater than the expression level of the corresponding endogenous genes in activated leukocytes (e.g., T cells, e.g., the expression level of the masking transgene is equal to the expression level of the endogenous gene in activated leukocytes, or is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more higher than the expression level of the endogenous gene in activated leukocytes). In some embodiments, all eight of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at levels equal to or greater than the expression levels of the corresponding endogenous genes in activated leukocytes.

[0057] In some embodiments of any of the foregoing aspects, one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level greater than the expression level of the corresponding endogenous genes in wild-type stem cells (e.g., wild-type ES cells from the same species, e.g., the expression level of the masking transgene in the masked cells is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1,000-fold or more higher than the expression of the endogenous gene in unmodified wild-type ES cells from the same species). In some embodiments, all eight of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at levels greater than (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100-fold or more) the expression level of endogenous genes in wild-type stem cells (e.g., embryonic stem cells from the same species as the masked cells). In some embodiments, one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level within the top 5% of gene expression of all genes in the ES cell genome. In some embodiments, one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level within the top 1% of gene expression of all genes in the ES cell genome. In some embodiments, PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are all expressed at levels within the top 5% of gene expression of all genes in the ES cell genome.

[0058] In some embodiments of any of the preceding aspects, the PD-L1 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity, or more) to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0059] In some embodiments of any of the preceding aspects, the HLA-G (H2-M3) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:15.

[0060] In some embodiments of any of the preceding aspects, the Cd47 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity, or more) to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0061] In some embodiments of any of the preceding aspects, the CD200 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0062] In some embodiments of any of the preceding aspects, the FASLG (FasL) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:9.

[0063] In some embodiments of any of the aforementioned aspects, the Ccl21 (Ccl21b) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1.

[0064] In some embodiments of any of the foregoing aspects, the Mfge8 transgene encodes a protein that has at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14.

[0065] In some embodiments of any of the aforementioned aspects, the Serpin B9 (Spi6) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:7.

[0066] In some embodiments of any of the preceding aspects, the IFNγR1 d39 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO:17.

[0067] In some embodiments of any of the foregoing aspects, the one or more transgenes are operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is selected from the group consisting of: CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, Epstein Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter.

[0068] In some embodiments of any one of the foregoing aspects, the cell further comprises (e.g., the cell is further modified to comprise) a transgene encoding a therapeutic agent. In some embodiments, the therapeutic agent is a protein or an antibody. In some embodiments, the antibody is an inhibitory antibody or an agonist antibody. In some embodiments, the therapeutic agent is an agent listed in Table 2. In some embodiments, the therapeutic agent is a wild-type version of a gene mutated in a subject (e.g., a wild-type version of a gene mutated in a subject's disease or illness, such as a genetic mutation associated with cancer, enzyme or hormone deficiency, metabolic disorder, or degenerative disease). In some embodiments, the therapeutic agent is expressed using an inducible expression system selected from the group consisting of: a tetracycline response element, a light-inducible system, a radiation genetic system, a coumarate switch-inducible system, an ecdysone-inducible system, a destabilizing domain system, or a ligand-reversible dimerization system. In some embodiments, the therapeutic agent is expressed using a constitutive promoter selected from the group consisting of: CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter.

[0069] In another aspect, a genetically modified cell population according to any of the above cells is provided.

[0070] In one aspect, a method for providing local immunosuppression at a transplantation site in an allogeneic host is provided. The method comprises transplanting a genetically modified cell as described above or a population of genetically modified cells as described above at a transplantation site in an allogeneic host.

[0071] In another aspect, the invention features a composition containing the cells of the invention. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0072] In another aspect, a kit is featured, comprising a cell of the invention or a pharmaceutical composition of the invention.

[0073] In another aspect, features are methods of treating a disease or disorder in a subject by administering to the subject a cell of the invention or a composition of the invention. In some embodiments, the disease or disorder is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, nervous system disease (such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS), enzyme or hormone deficiency, metabolic disorder (e.g., lysosomal storage disease, galactosemia, maple syrup urine disease, phenylketonuria, glycogen storage disease, Mitochondrial disorders, Friedreich's ataxia, peroxisomal disorders, disorders of metal metabolism or organic acidemias, autoimmune diseases (e.g., psoriasis, systemic lupus erythematosus, Graves' disease, inflammatory bowel disease, Addison's disease, Sjögren's syndrome), Hashimoto's thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infectious diseases, hemophilia, degenerative diseases (e.g., , Charcot-Marie-Doucet disease, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, cystic fibrosis, cytochrome C oxidase deficiency, Ehlers-Danlos syndrome, essential tremor, fibrodysplasia ossificans progressiva, infantile neuroaxonal dystrophy, keratoconus, keratoglobose, muscular dystrophy, neuronal ceroid lipofuscinosis, preexisting illness, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age The cell further comprises a transgene encoding a corresponding therapeutic agent listed in Table 2 or a wild-type version of a gene mutated in the subject (e.g., a wild-type version of a gene mutated associated with the disease or disorder of the subject, such as a genetic mutation associated with cancer, an enzyme or hormone deficiency, a metabolic disorder, or a degenerative disease). In some embodiments, the disease or condition is age-related macular degeneration (e.g., wet AMD) or a retinal dystrophy, and the therapeutic agent is a VEGF inhibitor (e.g., a soluble form of a VEGF receptor (e.g., soluble VEGFR-1 or NRP-1), platelet factor-4, prolactin, SPARC, a VEGF inhibitory antibody (e.g., bevacizumab or ranibizumab), or a soluble decoy receptor described in Holash et al., Proc Natl Acad Sci USA 99: 11383-11398, 2002, e.g., VEGF-Trap 亲本 , VEGF-Trap ΔB1 , VEGF-TrapΔB2 , VEGF-Trap R1R2 , such as aflibercept). In some embodiments, the disease or disorder is osteoarthritis or rheumatoid arthritis, and the therapeutic agent is an anti-inflammatory biologic (e.g., a TNFα inhibitor (e.g., adalimumab, etanercept, infliximab, golimumab, or certolizumab pegol), an interleukin-6 (IL6) receptor inhibitor (e.g., tocilizumab), an IL1 receptor inhibitor (e.g., anakinra), or another agent used to treat rheumatoid arthritis (e.g., abatacept, rituximab)). In some embodiments, the disease or disorder is diabetes (e.g., type 1 diabetes or type 2 diabetes), and the therapeutic agent is insulin. In some embodiments, the disease or disorder is hemophilia, and the therapeutic agent is factor VIII. In some embodiments, the disease or disorder is a metabolic deficiency, and the therapeutic agent is a transgene having a nucleic acid sequence of a wild-type version of a gene mutated in the subject or a transgene encoding an enzyme that is deficient in the subject.

[0074] In some embodiments of any of the foregoing aspects, prior to administration to a subject, the cells are differentiated into lineage-restricted cell types. In some embodiments, the disease or disorder is myocardial infarction, and the cells are differentiated into cardiomyocytes. In some embodiments, the disease or disorder is blindness, and the cells are differentiated into photoreceptor cells. In some embodiments, the disease or disorder is spinal cord injury, Parkinson's disease, Huntington's disease, or Alzheimer's disease, and the cells are differentiated into neurons. In some embodiments, the disease or disorder is multiple sclerosis, and the cells are differentiated into glial cells.

[0075] In some embodiments of any of the preceding aspects, the cells are administered (eg, injected or implanted) locally to a tissue or body part in need of the cells or therapeutic agent.

[0076] In some embodiments of any of the preceding aspects, the cells are administered intravenously, subcutaneously, intramuscularly, transdermally, intradermally, parenterally, intraarterially, intravascularly, or by perfusion.

[0077] In some embodiments of any of the preceding aspects, the cells are administered by subcutaneous injection to produce concealed subcutaneous tissue.

[0078] In some embodiments of any of the aforementioned aspects, the cells are administered as a tissue. In some embodiments, the tissue is administered with a gel, a biocompatible matrix, or a cell scaffold.

[0079] In some embodiments of any of the foregoing aspects, the cells are present at a density of 25,000 to 5,000,000,000 cells (e.g., 2.5 x 10 4 , 5x 10 4 , 7.5x 10 4 , 1x 10 5 , 2x10 5 , 3x 10 5 , 4x 10 5 , 6x 10 5 , 6x 10 5 , 7x10 5 , 8x 10 5 , 9x 10 5 , 1x10 6 , 2x 10 6 , 3x 10 6 , 4x 10 6 , 5x 10 6 , 6x 10 6 , 7x 10 6 , 8x 10 6 , 9x10 6 , 1x10 7 , 2x 10 7 , 3x 10 7 , 4x 10 7 , 5x 10 7 , 6x 10 7 , 7x 10 7 , 8x10 7 , 9x 10 7 , 1x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x10 8 , 8x 10 8 , 9x 10 8 , 1x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 or 5x 10 9 cells) were administered.

[0080] In some embodiments of any of the foregoing aspects, the cells are present at a density of 800,000,000 to 100,000,000,000 cells (e.g., 8 x 10 8 , 9x 108 , 1x 10 9 , 2x 10 9 , or 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x 10 9 , 7x10 9 , 8x 10 9 , 9x 10 9 , 1x 10 10 , 2x 10 10 , 3x 10 10 , 4x 10 10 , 5x 10 10 , 6x 10 10 , 7x 10 10 , 8x 10 10 , 9x10 10 or 1x 10 11 cells) were administered.

[0081] In some embodiments of any of the foregoing methods, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered prior to administering the cells. In some embodiments, the additional therapeutic agent is administered after administering the cells. In some embodiments, the additional therapeutic agent is administered simultaneously with administering the cells.In some embodiments, the additional therapeutic agent is an immunosuppressant, a disease-modifying antirheumatic drug (DMARD), a biologic response modifier (a type of DMARD), a corticosteroid or a nonsteroidal anti-inflammatory drug (NSAID), prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, tofacitinib, adalimumab, abatacept, anakinra, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab or tocilizumab, 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab, aminosalicylic acid Salt, antibiotics, antihistamines, anti-TNFα, azathioprine, belimumab, interferon beta, calcineurin inhibitors, certolizumab pegol, corticosteroids, cromolyn, cyclosporine A, cyclosporine, dimethyl fumarate, etanercept, fingolimod, fumarate esters, copaxone, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonists, long-acting beta2 agonists, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, probiotics, retinoids, salicylic acid, short-acting beta2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab or vedolizumab, bevacizumab monoclonal antibody, ranibizumab or aflibercept), photodynamic therapy, photocoagulation, carbidopa-levodopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase inhibitors, anticholinergics, amantadine, deep brain stimulation, anticoagulants, antiplatelet agents, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers, angiotensin receptor neprilysin inhibitors, beta blockers, combined alpha and beta blockers, calcium channel blockers, cholesterol-lowering drugs, niacin, cholesterol absorption inhibitors, digitalis preparations, diuretics, vasodilators, dual antiplatelet therapy, cardiac surgery, antiviral compounds, nucleoside analog reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors enzyme inhibitors (NNRTI), protease inhibitors, antibacterial compounds, antifungal compounds, antiparasitic compounds, insulin, sulfonylureas, biguanides, meglitinides, thiazolidinediones, DPP-4 inhibitors, SGLT2 inhibitors, alpha-glucosidase inhibitors, bile acid sequestrants, aspirin, dietary regimens, coagulation factors, desmopressin, clot-preserving drugs, fibrin sealants, physical therapy, coenzymes, bone marrow transplants, organ transplants, hemodialysis, hemofiltration, exchange transfusions, peritoneal dialysis, medium chain triglycerides, miglustat, enzyme replacement therapy, checkpoint inhibitors, chemotherapeutic drugs, biologic drugs, radiation therapy, cryotherapy, hyperthermia, surgical removal of tumor tissue, or anticancer vaccines.

[0082] In some embodiments of any of the foregoing methods, the method further comprises controlling the proliferation of cells. In some embodiments, the cell comprises an ALINK system, and the method of controlling proliferation comprises: i) allowing the proliferation of cells comprising the ALINK system by maintaining the cell comprising the ALINK system in the absence of an inducer of a negative selective marker; or ii) eliminating or inhibiting the proliferation of cells comprising the ALINK system by exposing the cell comprising the ALINK system to an inducer of a negative selective marker. In some embodiments, the cell comprises an EARC system, and the method of controlling cell proliferation comprises: i) allowing the proliferation of cells comprising the EARC system by exposing the cell comprising the EARC system to an inducer of a gene expression system based on an inducible activating factor; or ii) preventing or inhibiting the proliferation of cells comprising the EARC system by maintaining the cell comprising the EARC system in the absence of an inducer of a gene expression system based on an inducible activating factor.

[0083] In some embodiments of any of the foregoing methods, the cells are removed after treatment is complete. The removal of the cells can be performed surgically (e.g., removing transplanted tissue or organs, or removing obscured subcutaneous tissue) or by using an ALINK and / or EARC system. In some embodiments, one or more (e.g., one, two, three, four or more) ALINK and / or EAReC systems are used to eliminate all obscured cells.

[0084] In another aspect, the invention provides a cell of the invention or a composition of the invention for use in treating a disease or disorder in a subject in need thereof. In some embodiments, the disease or disorder is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, nervous system disease (such as Parkinson's disease, Alzheimer's disease, Huntington's disease and ALS), enzyme or hormone deficiency, metabolic disorder (e.g., lysosomal storage disease, galactosemia, maple syrup urine disease, phenylketonuria, glycogen storage disease, mitochondrial disorder, Friedrich's ataxia, peroxisomal disorder, metal metabolism disorder or organic acidemia, autoimmune disease (e.g., psoriasis, systemic lupus erythematosus, Graves' disease, inflammatory bowel disease, Addison's disease, Sjögren's syndrome), Hashimoto's thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infectious disease, hemophilia, degenerative disease (e.g., Charcot-Marie-Doyle disease, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, cystic fibrosis, cytochrome C oxidase deficiency, Ehlers-Danlos syndrome, essential tremor, fibrodysplasia ossificans progressiva, infantile neuroaxonal dystrophy, keratoconus, keratoglobose, muscular dystrophy, neuronal ceroid lipofuscinosis, pre-existing disease, progressive supranuclear palsy, Sandhoff's disease, spinal muscular atrophy, retinitis pigmentosa) or age-related disease (e.g., atherosclerosis, cardiovascular disease (e.g., angina, myocardial infarction), cataract, osteoporosis, or hypertension), or a disease or condition listed in Table 2.

[0085] In another aspect, the invention provides a cell of the invention or a composition of the invention for use in providing local immunosuppression at a transplantation site in an allogeneic host.

[0086] In some embodiments of any of the foregoing aspects, the cells comprise two of the following transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1 and HLA-G (H2-M3); PD-L1 and Cd47; PD-L1 and Cd200; PD-L1 and FASLG (FasL); PD-L1 and Ccl21 (Ccl21b); PD-L1 and Mfge8; PD-L1 and SerpinB9 (Spi6); HLA-G (H2-M3) and Cd47; HLA-G (H2-M3) and Cd200; HLA -G (H2-M3) and FASLG (FasL); HLA-G (H2-M3) and Ccl21 (Ccl21b); HLA-G (H2-M3) and Mfge8; HLA-G (H2-M3) and SerpinB9 (Spi6); Cd47 and Cd200; Cd47 and FASLG (FasL); Cd47 and Ccl21 (Ccl21b); Cd47 and Mfge8; Cd47 and Serpin B9 (Spi6); Cd200 and FASLG (FasL); Cd200 and Ccl21 (Ccl21b); Cd200 and Mfge8; Cd200 and Serpin B9 (Spi6); FASLG (FasL) and Ccl21 (Ccl21b); FASLG (FasL) and Mfge8; FASLG (FasL) and SerpinB9 (Spi6); Ccl21 (Ccl21b) and Mfge8; Ccl21 (Ccl21b) and Serpin B9 (Spi6); or Mfge8 and Serpin B9 (Spi6)).

[0087] In some embodiments of any of the foregoing aspects, the cells comprise three of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3) and Cd47; PD-L1; HLA-G (H2-M3) and Cd200; PD-L1, HLA-G (H2-M3) and FASLG (FasL); PD-L1, HLA-G (H2-M3) and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3) and Mfge8; PD-L1, HLA-G (H2-M3) and Serpin B9 (Spi6); PD-L1, Cd47 and Cd200; PD-L1, Cd47 and FASLG (FasL); PD-L1, Cd47 and Ccl21 (Ccl21b); PD-L1, Cd47 and Mfge8; PD-L1, Cd47 and Serpin B9; PD-L1, Cd200 and FASLG (FasL); PD-L1, Cd200 and Ccl21 (Ccl21b); PD-L1, Cd200 and Mfge8; PD-L1; Cd200 and Serpin B9 (Spi6); PD-L1, FASLG (FasL) and Ccl21 (Ccl21b); PD-L1, FASLG (FasL) and Mfge8; PD-L1, FASLG (FasL) and Serpin B9 (Spi6); PD-L1, Ccl21 (Ccl21b) and Mfge8; PD-L1, Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47 and Cd200; HLA-G (H2-M3), Cd47 and FASLG (FasL); HLA-G (H2-M3), Cd47 and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd47 and Mfge8; HLA-G (H2-M3), Cd47 and Serpin B9; HLA-G (H2-M3), Cd200 and FASLG (FasL); HLA-G (H2-M3), Cd200 and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd200 and Mfge8; HLA-G (H2-M3), Cd200 and Serpin B9; HLA-G (H2-M3), FASLG (FasL) and Ccl21 (Ccl21b); HLA-G (H2-M3), FASLG (FasL) and Mfge8;HLA-G (H2-M3), FASLG (FasL) and SerpinB9 (Spi6); HLA-G (H2-M3), Ccl21 (Ccl21b) and Mfge8; HLA-G (H2-M3), Ccl21 (Ccl21b) and Serpin B9 (Spi6); HLA-G (H2-M3), Mfge8 and SerpinB9 (Spi6); Cd47, Cd200 and FASLG (FasL); Cd47, Cd200 and Ccl21 (Ccl21b); Cd47, Cd200 and Mfge8; Cd47, Cd200 and Serpin B9 (Spi6); Cd47, FASLG (FasL) and Ccl21 (Ccl21b); Cd47, FASLG (FasL) and Mfge8; Cd47, FASLG (FasL) and Serpin B9 (Spi6); Cd47, Ccl21 (Ccl21b) and Mfge8; Cd47, Ccl21 (Ccl21b) and Serpin B9 (Spi6); Cd47, Mfge8 and Serpin B9 (Spi6); Cd200, FASLG (FasL) and Ccl21 (Ccl21b); Cd200, FASLG (FasL) and Mfge8; Cd200, FASLG (FasL) and Serpin B9 (Spi6); Cd200, Ccl21 (Ccl21b) and Mfge8; Cd200, Ccl21 (Ccl21b) and Serpin B9 (Spi6); Cd200, Mfge8 and Serpin B9 (Spi6); FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; FASLG (FasL), Ccl21 (Ccl21b) and Serpin B9 (Spi6); Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6)).

[0088] In some embodiments of any of the foregoing aspects, the cells comprise four of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), Cd47, and Cd200; PD-L1, HLA-G (H2-M3), Cd47, and FASLG (FasL); PD-L1, HLA-G (H2-M3), Cd47, and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200 and FASLG (FasL); PD-L1, HLA-G (H2-M3), Cd200 and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd200 and Mfge8; PD-L1, HLA-G (H2-M3), Cd200 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), FASLG (FasL), and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), FASLG (FasL), and Mfge8; PD-L1, HLA-G (H2-M3), FASLG (FasL), and SerpinB9 (Spi6); PD-L1, HLA-G (H2-M3), Ccl21 (Ccl21b), and Mfge8; PD-L1, HLA-G (H2-M3), Ccl21 (Ccl21b), and SerpinB9 (Spi6); PD-L1, HLA-G (H2-M3), Mfge8, and Serpin B9 (Spi6); PD-L1, Cd47, Cd200 and FASLG (FasL); PD-L1, Cd47, Cd200 and Ccl21 (Ccl21b); PD-L1, Cd47, Cd200 and Mfge8; PD-L1, Cd47, Cd200 and Serpin B9 (Spi6); PD-L1, Cd47, FASLG (FasL) and Ccl21 (Ccl21b); PD-L1, Cd47, FASLG (FasL) and Mfge8; PD-L1, Cd47, FASLG (FasL) and Serpin B9 (Spi6); PD-L1, Cd47, Ccl21 (Ccl21b) and Mfge8; PD-L1, Cd47, Ccl21 (Ccl21b) and Serpin B9(Spi6);PD-L1, Cd47, Mfge8, and Serpin B9 (Spi6); PD-L1, Cd200, FASLG (FasL), and Ccl21 (Ccl21b); PD-L1, Cd200, FASLG (FasL), and Mfge8; PD-L1, Cd200, FASLG (FasL), and Serpin B9 (Spi6); PD-L1, Cd200, Ccl21 (Ccl21b), and Mfge8; PD-L1, Cd200, Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, Cd200, Mfge8, and Serpin B9 (Spi6); PD-L1, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, FASLG (FasL), Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); PD-L1, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Cd200 and FASLG (FasL); HLA-G (H2-M3), Cd47, Cd200 and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd47, Cd200 and Mfge8; HLA-G (H2-M3), Cd47, Cd200 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, FASLG (FasL) and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd47, FASLG (FasL) and Mfge8; HLA-G (H2-M3), Cd47, FASLG (FasL) and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b) and Mfge8; HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b) and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd200, FASLG (FasL) and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd200, FASLG (FasL) and Mfge8; HLA-G (H2-M3), Cd200, FASLG (FasL) and Serpin B9 (Spi6); HLA-G (H2-M3), Cd200, Ccl21 (Ccl21b), and Mfge8;HLA-G (H2-M3), Cd200, Ccl21 (Ccl21b), and Serpin B9 (Spi6); HLA-G (H2-M3), Cd200, Mfge8, and Serpin B9 (Spi6); HLA-G (H2-M3), FASLG (FasL), Ccl21 (Ccl21b), and Mfge8; HLA-G (H2-M3), FASLG (FasL), Ccl21 (Ccl21b), and Serpin B9 (Spi6); HLA-G (H2-M3), FASLG (FasL), Mfge8, and Serpin B9 (Spi6); B9 (Spi6); Cd47, Cd200, FASLG (FasL) and Ccl21 (Ccl21b); Cd47, Cd200, FASLG (FasL) and Mfge8; Cd47, Cd200, FASLG (FasL) and Serpin B9 (Spi6); Cd47, Cd200, Ccl21 (Ccl21b) and Mfge8; Cd47, Cd200, Ccl21 (Ccl21b) and Serpin B9 (Spi6); Cd47, Cd200, Mfge8 and Serpin B9 (Spi6); Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Serpin B9 (Spi6); Cd47, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); Cd200, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; Cd200, FASLG (FasL), Ccl21 (Ccl21b) and SerpinB9 (Spi6); Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); or FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6)). ;

[0089] In some embodiments of any of the foregoing aspects, the cell comprises five of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), Cd47, Cd200, and FASLG (FasL); PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Mfge8, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL) and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL) and Mfge8, PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL) and SerpinB9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, Ccl21 (Ccl21b) and SerpinB9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, Mfge8 and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), FASLG(FasL), Ccl21(Ccl21b) and Mfge8; PD-L1, HLA-G(H2-M3), FASLG(FasL), Ccl21(Ccl21b) and SerpinB9(Spi6);PD-L1, HLA-G (H2-M3), FASLG (FasL), Mfge8, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Ccl21 (Ccl21b), Mfge8, and SerpinB9 (Spi6); PD-L1, Cd47, Cd200, FASLG (FasL), and Ccl21 (Ccl21b); PD-L1, Cd47, Cd200, FASLG (FasL), and Mfge8, PD-L1, Cd47, Cd200, FASLG (FasL), and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, Ccl21 (Ccl21b) and Mfge8; PD-L1, Cd47, Cd200, Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, Mfge8 and Serpin B9 (Spi6); PD-L1, Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, Cd47, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); PD-L1, Cd47, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6); PD-L1, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Mfge8; PD-L1, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, Cd200, FASLG (FasL), Mfge8, and Serpin B9 (Spi6); PD-L1, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6); PD-L1, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL) and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL) and Mfge8; HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL) and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b) and Mfge8;HLA-G(H2-M3), Cd47, Cd200, Ccl21(Ccl21b) and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, Cd200, Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, FASLG(FasL), Ccl21(Ccl21b) and Mfge8; HLA-G(H2-M3), Cd47, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, FASLG(FasL), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Cd200, FASLG(FasL), Ccl21(Ccl21b) and Mfge8; HLA-G(H2-M3), Cd200, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); HLA-G(H2-M3), Cd200, FASLG(FasL), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Cd200, Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), FASLG(FasL), Ccl21(Ccl21b), Mfge8 and Serpin B9 (Spi6); Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b) and Serpin B9 (Spi6); Cd47, Cd200, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); Cd47, Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); Cd47, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6);or Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6)). In some embodiments of any of the foregoing aspects, the cell comprises six of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b) and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd47, FASLG(FasL), Mfge8 and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd200, FASLG(FasL), Ccl21(Ccl21b) and Mfge8; PD-L1, HLA-G(H2-M3), Cd200, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd200, FASLG(FasL), Mfge8 and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd200, Ccl21(Ccl21b), Mfge8 and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Mfge8;PD-L1, Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, FASLG (FasL), Mfge8, and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6); PD-L1, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b) and Mfge8; HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, Cd200, Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, FASLG(FasL), Ccl21(Ccl21b), Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); or Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6)). ;

[0090] In some embodiments of any of the foregoing aspects, the cell comprises seven of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Serpin B9 (Spi6); B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); or HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6).

[0091] In some embodiments of any of the preceding aspects, the cells comprise all eight of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0092] In some embodiments of any of the foregoing aspects, the cells comprise one or more (e.g., one, two, three, four, five, six, or all seven) of the following group of transgenes: HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0093] In some embodiments of any of the aforementioned aspects, the cells comprise one or more (e.g., one, two, three, four, five, six, or all seven) of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0094] In some embodiments of any of the foregoing aspects, the cells comprise one or more (e.g., one, two, three, four, five, or all six) of the following group of transgenes: HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0095] In some embodiments of any of the preceding aspects, the cells are not modified to express PD-L1.

[0096] In some embodiments of any of the preceding aspects, the cell is not modified to express FasL.

[0097] In some embodiments of any of the preceding aspects, the cell is not modified to express TGF-β. In some embodiments of any of the preceding aspects, the cell is not modified to express CTLA4 or CLTA4-Ig. In some embodiments of any of the preceding aspects, the cell is not modified to express IDO. In some embodiments of any of the preceding aspects, the cell is not modified to express IL-35. In some embodiments of any of the preceding aspects, the cell is not modified to express IL-10. In some embodiments of any of the preceding aspects, the cell is not modified to express VEGF. In some embodiments of any of the preceding aspects, the cell is not modified to express NFkb decoy receptor. In some embodiments of any of the preceding aspects, the cell is not modified to express soluble TNFR. In some embodiments of any of the preceding aspects, the cell is not modified to express CCR7. In some embodiments of any of the preceding aspects, the cell is not modified to express SOCS1. In some embodiments of any of the preceding aspects, the cell is not modified to express HLA-E. In some embodiments of any of the aforementioned aspects, the cells are not modified to express siRNA to IL-12.

[0098] definition

[0099] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0100] As used herein, the term "about" refers to a value that is no more than 10% above or below the described value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0101] As used herein, the term "activated leukocytes" refers to the state of leukocytes (e.g., granulocytes, such as neutrophils, eosinophils or basophils; monocytes or lymphocytes, such as B or T cells) caused by a response to perceived damage. When leukocytes become activated, they can proliferate, secrete cytokines, differentiate, present antigens, become more polarized, become more phagocytic and / or become more cytotoxic. Factors that stimulate immune cell activation include proinflammatory cytokines, pathogens, and non-self antigen presentation. Activated leukocytes can be separated from lymphoid organs. Anti-CD3 / CD28 beads or other methods used by those skilled in the art can also be used to activate leukocytes in vitro, such as T cells (see, e.g., Frauwith and Thompson, J. Clin Invest 109: 295-299 (2002); and Trickett and Kwan, J Immunol Methods 275: 251-255 (2003)).

[0102] As used herein, "allogeneic" refers to cells, tissues, DNA or factors taken or derived from a different subject of the same species.

[0103] As used herein, the term "stem cell" refers to a cell that can be differentiated into one or more specialized cells and has self-renewal ability. Stem cells include pluripotent stem cells (PSC), such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC); and multipotent stem cells, such as cord blood stem cells, mesenchymal stromal cells and adult stem cells, which are found in various tissues. The term "stem cell" also includes cells suitable for genome editing, cells that can serve as sources of therapeutic cell types (e.g., cells that can be directed to differentiate into lineage-restricted or terminally differentiated cells for cell therapy, or cells of desired target tissues) and cells with stem cell properties (e.g., pluripotency or multipotency or self-renewal) obtained by "artificial" cells.

[0104] As used herein, the terms "embryonic stem cells" and "ES cells" refer to omnipotent or pluripotent stem cells derived from the embryonic origin of the inner cell mass of the blastocyst that can be maintained in in vitro culture under suitable conditions. ES cells are capable of differentiating into cells of any of the three vertebrate germ layers (e.g., endoderm, ectoderm, or mesoderm). ES cells are also characterized by their ability to propagate indefinitely under suitable in vitro culture conditions. See, for example, Thomson et al., Science 282: 1145 (1998).

[0105] As used herein, the terms "induced pluripotent stem cells", "iPS cells" and "iPSCs" refer to pluripotent stem cells that can be directly derived from differentiated somatic cells. Human iPS cells can be produced by introducing a specific set of reprogramming factors into non-pluripotent cells, and the non-pluripotent cells may include, for example, Oct3 / 4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, 1-Myc, n-Myc), Kruppel-like family (KLF) transcription factors (e.g., KLF1, KLF2, KLF4, KLF5) and / or related transcription factors, such as NANOG, LIN28 and / or Glis1. Human iPS cells can also be produced, for example, by using miRNA, small molecules that mimic the effects of transcription factors, or lineage-specific molecules (lineage specifier). Human iPS cells are characterized in that they can differentiate into any cell of the three vertebrate germ layers (e.g., endoderm, ectoderm or mesoderm). Human iPS cells are also characterized by their ability to propagate indefinitely under suitable in vitro culture conditions. See, e.g., Takahashi and Yamanaka, Cell 126:663 (2006).

[0106] As used herein, the term "reducing antigen presenting cell activation and function" refers to a transgene encoding a gene product whose function is to inhibit antigen presenting cell activation or the ability of antigen presenting cells to promote the activation of leukocytes that attack the transplant (Fiorentino et al., J Immunol. 146:3444-51 (1991); Salio et al., Eur J Immunol. 29:3245-53 (1999)). In one embodiment, reduction in antigen presenting cell activation and function refers to a decrease in APC activation and function relative to a control by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% (e.g., as determined using an assay for antigen presenting cell activation, such as decreased proliferation, expression of proinflammatory cytokines (e.g., interleukin-1 (IL-1, e.g., IL-1β), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF , such as TNFα), interferon gamma (IFNγ) and granulocyte macrophage colony stimulating factor (GMCSF), which can be measured using ELISA or Western blot analysis of culture medium or patient samples (such as blood samples), or cell surface markers (e.g., CD11c, CD11b, HLA molecules (e.g., MHC-II), CD40, B7, IL-2, CD80 or CD86, which can be assessed using flow cytometry, immunohistochemistry, in situ hybridization and other assays that allow measurement of cell surface markers)). Antigen presenting cells include dendritic cells, B cells and macrophages. Mast cells and neutrophils can also be induced to present antigens. Methods for determining reduction of antigen presenting cell activation and function are known in the art. Examples of gene products that reduce antigen presenting cell activation and function include, but are not limited to: Ccl21 (Ccl21b) and PD-L1. Such transgenes may be referred to herein as "masked" or "masked" genes.

[0107] As used herein, the term "reducing the activity of leukocytes attacking a graft or cytolytic function" refers to a transgene encoding a gene product whose function is to inhibit or prevent the activity of leukocytes attacking a graft or cytolytic function in the vicinity of allogeneic transplanted cells (MacDonald et al., J Immunol. 126: 1671-5 (1981); Bongrand et al., Eur J Immunol. 13: 424-9 (1983); MacDonald et al., Eur J Immunol. 9: 466-70 (1979)). In one embodiment, reduction in leukocyte activity or cytolytic function of the attacking transplant refers to a decrease in leukocyte activity or cytolytic function relative to a control by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% (e.g., as determined using an assay for leukocyte activation, such as decreased proliferation, proinflammatory cytokines (e.g., interleukin-1 (IL-1, e.g., IL-1β), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor ( TNF, such as TNFα), interferon gamma (IFNγ), and granulocyte macrophage colony stimulating factor (GMCSF), which can be measured using ELISA or Western blot analysis of culture medium or patient samples (such as blood samples), or polarization (e.g., decreased levels of IL-12, TNF, IL-1β, IL-6, IL-23, MARCO, MHC-II, CD86, iNOS, CXCL9, and CXCL10 in macrophages or monocytes, or Th1-specific markers in T cells (e.g., T-bet, IL-1β, IL-6, IL-23, MARCO, MHC-II, CD86, iNOS, CXCL9, and CXCL10 in T cells). 2R, STAT4), chemokine receptors (e.g., CCR5, CXCR6, or CXCR3), or Th2-specific markers: (e.g., CCR3, CXCR4, STAT6, GATA3, or IL-4Rα), which can be assessed using flow cytometry, immunohistochemistry, in situ hybridization, qPCR, or Western blot analysis for cellular markers or intracellular proteins, and ELISA or Western analysis for secreted proteins); or as determined using an assay for cytolytic function (e.g., by comparing leukocytes with cells pre-coated with The target cell line is incubated with an antibody against a surface antigen expressed by the target cell line and the number of viable target cells is measured with fluorescent viability staining or by measuring the secretion of cytolytic granules from leukocytes (e.g., perforin, granzyme, or other cytolytic proteins released from immune cells). Methods for determining the reduction of leukocyte activity or cytolytic function that attacks the transplant are known in the art. Examples of gene products that reduce leukocyte activity or cytolytic function that attacks the transplant include, but are not limited to: PD-L1, HLA-G (H2-M3), Cd39, Cd73, and Lag3.Such transgenes may be referred to herein as "masked" or "masked" genes.

[0108] As used herein, the term "reducing macrophage lytic function and phagocytosis of allogeneic transplant cells" refers to a transgene encoding a gene product whose function is to inhibit or prevent macrophage lytic function and / or phagocytosis of allogeneic transplant cells (Fish et al., Toxicology. 19: 127-38. (1981); Sung et al., J Biol Chem. 260: 546-54 (1985); Amash et al., J Immunol. 196: 3331-40 (2016)). In one embodiment, reduction in macrophage cytolytic function and phagocytosis of allogeneic transplant cells refers to a reduction in macrophage cytolytic function and / or phagocytosis of allogeneic transplant cells relative to a control by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% (e.g., as determined using an assay for macrophage cytolytic function (e.g., by incubating macrophages with a target cell line that has been pre-coated with antibodies to surface antigens expressed by the target cell line and measuring the number of viable target cells with fluorescent viability staining or by measuring the release of cytolytic granules (e.g., perforin, granzyme) from the macrophages). or other cytolytic proteins released from immune cells); or as determined using an assay for macrophage phagocytosis (e.g., culturing macrophages with fluorescent beads or a target cell line that has been pre-coated with antibodies to surface antigens expressed by the target cell line and measuring fluorescence inside the immune cells or quantifying the number of engulfed beads or cells). Methods for determining reduction of macrophage cytolytic function and phagocytosis of allogeneic graft cells are known in the art. Examples of gene products that reduce macrophage cytolytic function include, but are not limited to: Cd47, Cd200, Mfge8, and Il1r2. Such transgenes may be referred to herein as "masked" or "masked" genes.

[0109] As used herein, the term "inducing apoptosis of leukocytes attacking the graft" refers to a transgene encoding a gene product whose function is to kill leukocytes attacking the graft near allogeneic graft cells (Huang et al., Proc Natl Acad Sci USA. 96: 14871-6 (1999); Suzuki et al., Proc Natl Acad Sci USA. 97: 1707-12 (2000); Simon et al., Proc Natl Acad Sci USA. 98: 5158-63 (2001)). In one embodiment, induction of apoptosis of leukocytes attacking the graft refers to an increase in apoptosis of leukocytes attacking the graft by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control (e.g., as determined using an assay for apoptosis such as TUNEL staining, caspase staining or annexin-V staining or using fluorescent viability staining). Methods for determining the induction of apoptosis of leukocytes attacking the transplant are known in the art. Examples of gene products that can induce apoptosis of leukocytes attacking the transplant include, but are not limited to, FASLG (FasL) and Tnfsf10. Such transgenes may be referred to herein as "masked" or "masked" genes.

[0110] As used herein, the term "reducing local inflammatory proteins" refers to a transgene encoding a gene product whose function is to inhibit the activity of a local protein, wherein the function of the protein is to promote the accumulation of leukocytes attacking the transplant and / or their cytolytic function (Felix et al., Nat Rev Immunol. 17: 112-29 (2017)). In one embodiment, reduction of local inflammatory proteins refers to a reduction of local inflammatory proteins by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control (e.g., as determined using an assay for an inflammatory protein that promotes leukocyte activation or migration to the site of inflammation (e.g., chemokines such as CCL2, CCL3, CCL5, CXCL1, CXCL2, and CXCL8 or proinflammatory cytokines such as IL-1β, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, TNFα, IFNγ, or GMCSF, which can be measured using ELISA, Western blot analysis, or other techniques known in the art for measuring secreted proteins). Methods for determining reduction of local inflammatory proteins are known in the art. Examples of gene products that reduce local inflammatory proteins include, but are not limited to, PD-L1, Il1r2, and Ackr2. Such transgenes may be referred to herein as "masked" or "masked" genes.

[0111] As used herein, the term "protection from leukocyte-mediated apoptosis" refers to a transgene encoding a gene product that functions to inhibit any cellular component that can induce apoptosis or cytolysis of allogeneic graft cells (Abdullah et al., J Immunol. 178:3390-9 (2007)). In one embodiment, protection from leukocyte-mediated apoptosis refers to a reduction in leukocyte-mediated apoptosis of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control (e.g., as determined using an assay for leukocyte-mediated apoptosis (e.g., by incubating leukocytes with a target cell line that has been pre-coated with antibodies to a surface antigen expressed by the target cell line and measuring the number of viable target cells with fluorescent viability staining or by measuring the secretion of cytolytic granules released from the leukocytes (e.g., perforin, granzymes, or other cytolytic proteins released from immune cells)). Methods for determining protection from leukocyte-mediated apoptosis are known in the art. Examples of gene products that protect against leukocyte-mediated apoptosis include, but are not limited to, Serpin B9 (Spi6) and Dad1. Such transgenes may be referred to herein as "masked" or "masked" genes.

[0112] As used herein, the term "biological agent" refers to a designed polypeptide and corresponding coding DNA that can be expressed as a transgenic. The polypeptide can excite or inhibit the function of an endogenous gene or inhibit or activate a biological process. Methods for determining whether a polypeptide has agonist or antagonist activity or function are generally known in the art. In one embodiment, the agonist function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the function relative to the control. In one embodiment, the antagonist function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the function relative to the control.

[0113] As used herein, the term "operably connected" refers to that a first molecule is connected to a second molecule, wherein the molecule is arranged so that the first molecule affects the function or expression of the second molecule. Two molecules may or may not be a part of a single adjacent molecule and may or may not be adjacent. For example, if the promoter regulates the transcription of the target transcribable polynucleotide molecule in the cell, the promoter is operably connected to the transcribable polynucleotide molecule. In addition, if the two parts of the transcriptional regulatory element are linked together so that the transcriptional activation functionality of one part is not adversely affected by the presence of another part, the two parts are operably connected to each other. Two transcriptional regulatory elements can be operably connected to each other by a joint nucleic acid (for example, inserting a non-coding nucleic acid), or can be operably connected to each other in the absence of an inserted nucleotide.

[0114] As used herein, the term "promoter" refers to a recognition site on DNA to which RNA polymerase binds. The polymerase drives transcription of a transgene.

[0115] " Percentage (%) of sequence identity " with respect to reference polynucleotide or peptide sequence is defined as after comparing sequence and introducing room to realize maximum sequence identity percentage when necessary, the percentage of nucleic acid or amino acid identical with the nucleic acid in reference polynucleotide or peptide sequence in candidate sequence.Comparison for measuring nucleic acid or amino acid sequence identity percentage purpose can be realized in a variety of ways within the capabilities of those skilled in the art, and the mode for example uses publicly available computer software, such as BLAST, BLAST-2 or Megalign software.Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for maximum comparison on the total length of the sequence compared.For example, sequence comparison computer program BLAST can be used to produce sequence identity percentage value.As an illustration, given nucleic acid or amino acid sequence A for, with or for given nucleic acid or amino acid sequence B sequence identity percentage (it can be alternatively phrased as given nucleic acid or amino acid sequence A has or comprises for, with or for given nucleic acid or amino acid sequence B a certain sequence identity percentage) is calculated as follows:

[0116] 100×(fraction X / Y)

[0117] wherein X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in the program's alignment of A and B, and wherein Y is the total number of nucleic acids in B. It will be understood that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0118] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic agents, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and / or carriers, to be administered to a subject, such as a mammal (e.g., a human) to prevent, treat or control a particular disease or condition that affects or may affect the subject.

[0119] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of subjects, such as mammals (e.g., humans) without excessive toxicity, irritation, allergic response and / or other problematic complications, commensurate with a reasonable benefit / risk ratio.

[0120] As used herein, the term "wild type" refers to the genotype with the highest frequency of a particular gene in a given organism.

[0121] As used herein, the terms "cell division locus", "cell division loci" and "CDL" refer to a genomic locus (or loci) whose transcription products are expressed by dividing cells. When the CDL comprises a single locus, the absence of CDL expression in a cell (or its derivative) means that tumor initiation and / or formation is prohibited because the cell will be eliminated in the absence of CDL expression or because the proliferation of the cell will be blocked or impaired in the absence of CDL expression. When the CDL comprises multiple loci, the absence of expression of all or a subset of the loci in a cell (or its derivative) means that tumor initiation and / or formation is prohibited because the cell will be eliminated in the absence of CDL expression or because the proliferation of the cell will be blocked or impaired in the absence of CDL expression. The CDL may or may not be expressed in non-dividing and / or non-proliferating cells. The CDL may be endogenous to the host cell, or it may be a transgenic. If the CDL is a transgenic, it may be from the same or different species as the host cell, or it may have a synthetic origin. In one embodiment, the CDL is a single locus transcribed during cell division. For example, in one embodiment, a single locus CDL is CDK1. In one embodiment, a CDL comprises two or more loci that are transcribed during cell division. For example, in one embodiment, a multi-locus CDL comprises two MYC genes (c-Myc and N-myc) (Scognamiglio et al., 2016). In one embodiment, a multi-locus CDL comprises AURORA B and C kinases that may have overlapping functions (Fernandez-Miranda et al., 2011). Cell division and cell proliferation are terms that may be used interchangeably herein.

[0122] As used herein, the terms "normal rate of cell division," "normal cell division rate," "normal rate of cell proliferation," and "normal cell proliferation rate" refer to the rate of cell division and / or proliferation that is typical of non-cancerous healthy cells. The normal rate of cell division and / or proliferation can be cell type specific. For example, it is well known that the number of cells in the epidermis, intestine, lung, blood, bone marrow, thymus, testis, uterus, and breast is maintained by a high rate of cell division and a high rate of cell death. In contrast, the number of cells in the pancreas, kidney, cornea, prostate, bone, heart, and brain is maintained by a low rate of cell division and a low rate of cell death (Pellettieri and Sánchez Alvarado, 2007).

[0123] As used herein, the terms "inducible negative effector of proliferation" and "iNEP" refer to genetic modifications that promote the use of CDL expression to control cell division and / or proliferation by: i) inducibly terminating or blocking CDL expression, thereby prohibiting cell division and proliferation; ii) inducibly eliminating at least a portion of cells expressing CDL (i.e., killing at least a portion of proliferating cells); or iii) inducibly slowing the rate of cell division relative to the normal cell division rate of the cell, so that the cell division rate will not be fast enough to promote tumor formation.

[0124] As used herein, the terms "elimination linkage" and "ALINK" refer to examples of iNEPs that include a transcriptional linkage between a CDL and a sequence encoding a negatively selective marker. ALINK modification allows the user to inductively kill proliferating host cells comprising ALINK or inhibit the proliferation of host cells by exposing ALINK-modified cells to an inducer of a negatively selective marker to kill at least a portion of the proliferating cells. For example, cells modified to include ALINK at the CDL can be treated with an inducer of a negatively selective marker (e.g., a prodrug) to eliminate proliferating cells or inhibit cell proliferation by killing at least a portion of the proliferating cells.

[0125] As used herein, the terms "exogenous activating factors regulated by CDL" and "EARC" refer to examples of iNEPs, which include mechanisms or systems that promote exogenous changes in transcription of non-coding or coding DNA or corresponding translation via activating factors. EARC modification allows the user to inductively terminate or inhibit the division of cells containing EARC by removing the inducer that allows transcription and / or translation of EARC-modified CDL from the EARC-modified cells. For example, a gene expression system based on an inducible activating factor can be operably connected to a CDL and used to exogenously control the expression of CDL or CDL translation, so that the presence of a drug-inducible activating factor and a corresponding inducer drug is necessary for CDL transcription and / or translation. In the absence of an inducer drug, cell division and / or proliferation will terminate or be inhibited (e.g., slowed to a normal cell division rate). For example, the CDL Cdk1 / CDK1 can be modified to include a dox-bridge so that the expression of Cdk1 / CDK1 and cell division and proliferation are only possible in the presence of an inducer (e.g., doxycycline).

[0126] As used herein, the term "proliferation antagonist system" refers to a natural or engineered compound whose presence inhibits (completely or partially) the proliferation of a cell.

[0127] As used herein, the term "dox-bridge" refers to a mechanism that separates the activity of the promoter from the target transcription region and realizes the transcription of the target region under the control of TRE by expressing rtTA (Gossen et al., 1995) from an endogenous or exogenous promoter. As used herein, "rtTA" refers to the reverse tetracycline transactivator element (Gossen et al., 1995) of the tetracycline inducible system, and "TRE" refers to a promoter consisting of the TetO operon sequence upstream of the minimal promoter. After the binding of rtTA to the TRE promoter in the presence of doxycycline, the transcription of the locus downstream of the TRE promoter increases. The rtTA sequence can be inserted into the same transcription unit as CDL, or into different locations of the genome, as long as the permissible or nonpermissible state of the transcriptional expression of the target region is controlled by doxycycline. Dox-bridge is an example of EARC.

[0128] As used herein, the term "fail-safe cell" refers to a cell containing one or more homozygous, heterozygous, hemizygous, or compound heterozygous ALINK or EARC in one or more CDLs (e.g., at least two, three, four, or five CDLs). A fail-safe cell may contain ALINK or EARC or both ALINK and EARC modifications (e.g., ALINK and EARC modifications in different CDLs or a single CDL).

[0129] As used herein, the term "fail-safe" refers to the property of cells that are less likely to exhibit uncontrolled (e.g., tumorigenic) proliferation. When cell proliferation is under the control of negative regulators or inducers and the probability of cell loss of activity of the system controlling proliferation due to genetic mutations is low, the cell can be considered "fail-safe". The fail-safe capacity will depend on the number of ALINKs and the number of CDLs targeted by ALINKs (e.g., cells with homozygous modifications of two different CDLs have a higher fail-safe capacity than cells with heterozygous modifications of a single CDL (e.g., less likely to lose all systems controlling proliferation through genetic mutations)). The fail-safe properties are further described in Table 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0131] These and other features of the present disclosure will become more apparent in the following detailed description taken in conjunction with the accompanying drawings, in which:

[0132] Figures 1A-1D Depicts the use of immunohistochemistry to mask proteins (Cd200 ( Figure 1A )、FasL( Figure 1B )、H2-M3( Figure 1C ) and Cd47( Figure 1D )) Representative images of the expression of .

[0133] Figure 2A-2E is to show the use of spleen cells in mixed lymphocyte reactions ( Figure 2A ), wt B16 melanoma cells ( Figure 2B ), masked B16 melanoma cells ( Figure 2C ), wt ES cells ( Figure 2D ) and masked ES cells ( Figure 2E ) of T cell activation.

[0134] Figure 3A-3B are shown with their WT counterparts ( Figure 3A ) compared to the masked ( Figure 3B ) Schematic diagram and images of B16F10 cancer cells protected from rejection. Unmasked cells in C57BL / 6 (n=5) and unmasked cells in FVB / N (n=4) ( Figure 3A ); cells masked in C57BL / 6 (n=5) and cells masked in FVB / N (n=6) ( Figure 3B ) representative images.

[0135] Figure 4 Schematic diagram showing that masked embryonic stem cells form tumors in isogenic B6 mouse recipients (upper panel) and FVB allogeneic recipients (lower panel).

[0136] Figures 5A-5C is a series of photographs depicting allogeneic mice bearing teratomas formed by subcutaneous injection of masked C57BL / 6 ES cells. The red arrows indicate the teratomas. Figure 5A shows a teratoma in a C3H mouse, Figure 5B shows teratoma in FVB / N mice, Figure 5C Shown is a teratoma in a CD1 mouse.

[0137] Figure 6 are schematic diagrams and series of images showing that animals with obscured tissue are not immunocompromised.

[0138] Figure 7 is a series of images of FVB / N mice showing additional results demonstrating that animals with obscured tissue are not immunocompromised.

[0139] Figures 8A-8H Transgene expression is shown in embryonic stem cells derived from C57BL / 6 mice containing clone safety knockout. Fig. 8A FasL expression is shown. Figure 8B Ccl21b expression is shown. Figure 8C Shows Cd200 expression, Fig.8D Showing Cd47 expression, Fig. 8E Mfge8 expression is shown. Fig.8F Spi6 expression is shown. Figure 8G H2-M3 expression is shown, and Figure 8H PD-L1 expression is shown.

[0140] Fig. 9 It is a series of figures describing the masked transgenic expression in ES cell clones. Each masked transgenic is depicted in different colors. Concentric circles represent the expression level by log10 scale. Thick black circles represent the 1x expression normalized to the positive control (activated leukocytes isolated from mouse lymphoid organs), wherein the next outer ring represents the 10x and 100x expression compared with the positive control, respectively. The innermost ring is the 0.1x expression compared with the positive control. Clone NT2 and 15 (represented by red squares) have the highest masked gene expression. These clones survive in allogeneic hosts.

[0141] Fig.10is a graph depicting the expression of masking transgenes in the genome-wide gene expression level distribution of the entire genome of ES cells. The expression levels of all eight masking transgenes in the NT2 cell line and NT2-derived teratomas are in the top 5% of all genes in the ES cell genome, with five masking transgenes having expression levels in the top 1% of all genes in the ES cell genome. The expression levels of the transgenes in the NT2 line and NT2-derived teratomas successfully achieve allograft tolerance.

[0142] Figures 11A-11B is a photograph showing C57BL / 6-derived teratomas in FVB / N mice. Transgenic line NT2 produced 9 teratomas in 10 injection sites. Images were taken 3 months after injection. Fig. 11B Is Fig.11A The arrows indicate magnified images of teratomas.

[0143] Figures 12A-12B is shown in the isogenic ( Fig. 12A ) and allogeneic ( Fig. 12B ) Graph of teratoma tumor size in mice.

[0144] Figures 13A-13B is shown to be injected with the same gene ( Fig.13A ) and allogeneic ( Fig. 13B A series of micrographs showing that cloaked embryonic stem cells in a ) host can differentiate into all three cell lineages.

[0145] Figures 14A-14D are photomicrographs showing the formation of all three germ layers in teratomas formed by subcutaneous injection of masked ES cells into mice. Fig.14A , Fig. 14B and Fig. 14C The three germ layers are shown (ec = ectoderm, Fig.14A shown in; en = endoderm, Fig. 14C shown in ; me = mesoderm, Fig. 14B as shown in . Fig.14D Blood vessels indicated by red arrows are shown, confirming that the tissue is well vascularized.

[0146] Fig.15 is a schematic diagram showing the construction of a vector expressing a target gene essential for allo-tolerance.

[0147] Figures 16A-16H are fluorescence micrographs showing expression of the protein encoded by the masking transgene in ES cells. Fig.16A The expression of PD-L1 is shown. Fig. 16B The expression of CD200 is shown. Fig. 16C The expression of CD47 is shown. Fig.16DThe expression of FasL is shown. Fig.16E The expression of H2-M3 is shown. Fig.16F The expression of Ccl21 is shown. Figure 16G shows the expression of Mfge8, and Fig.16H The expression of Spi6 is shown.

[0148] Figures 17A-17B The masked ES cells have typical ES cell morphology ( Fig.17A ) and expressed the ES cell marker alkaline phosphatase ( Fig. 17B ) micrograph.

[0149] Figures 18A-18B The pluripotent ES cell marker (Oct4 ( Fig.18A ) and SSEA1( Fig.18B ))'s fluorescence micrograph of the expression of . Figures 18A-18B The inset in shows single channel images of fluorescence micrographs of ES cell markers (Oct4 and SSEA) and DAPI labeling the nuclei to demonstrate that staining of ES cell markers co-localizes with the obscured cells.

[0150] Fig.19 Schematic diagram depicting the immune process of suppression in ES cells by masking a transgene (upper panel) and an expression cassette for expressing the masked transgene (lower panel).

[0151] Fig. 20 is a series of graphs depicting the effects of interferon gamma (IFNγ) on MHC levels in ES cells. IFNγ increases MHC levels in wild-type ES cells and in ES cells overexpressing the wild-type IFNγ receptor IFNγR1, but not in ES cells overexpressing a dominant negative form of the IFNγ receptor (IFNγR1 d39), indicating that IFNγR1 d39 completely inhibits IFNγ-mediated upregulation of MHC in ES cells. DETAILED DESCRIPTION

[0152] Description of cells and methods

[0153] Features are tools such as genetically modified cells and methods for using the cells to provide local immunosuppression at the site of transplantation, for example when the cells are transplanted in an allogeneic host. The genetically modified cells contain: one or a group of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane-bound or locally acting and whose function is to reduce the function of the host immune system (e.g., leukocytes attacking the transplant and NK cell activation) or serve as a defense mechanism against attacking leukocytes.

[0154] Various cytoplasmic, membrane-bound or locally acting immune factors have been found to regulate local immune compartments and local immune populations. Immune factors such as PD-L1 (Brown et al., J Immunol. 170: 1257-66 (2003; Curiel et al., Nat Med. 9: 562-7 (2003); Dong et al., Nat Med. 8: 793-800 (2002)), CD47 (Willingham et al., Proc Natl Acad Sci USA. 109: 6662-7 (2012); Liu et al., PLoS One.10:e0137345 (2015); Demeure et al., J Immunol.164:2193-9 (2000)), CD200 (Jenmalm et al., J Immunol.176:191-9 (2006); Cherwinski et al., J Immunol.174:1348-56 (2005); Kretz-Rommel et al., J Immunol.178:5595-605 (2007)), FasL (O'Connell et al., J Exp Med.184:1075-82 (1996); Ju et al., Nature.373:444-8 (1995); Mazar et al., J Biol Chem.284:22022-8 (2009)), and Spi6 (Medema Proceedings of the National Academy of of Sciences of the United States of America. 98: 11515-20 (2001); Zhang et al., Immunity. 24: 451-61 (2006); Soriano et al., Lung Cancer. 77: 38-45 (2012)) have been described in many, including their role in immunomodulation. It was found that the expression of one or more of these immunomodulatory factors in allogeneic cells can be used to provide local immunosuppression and / or reduce allogeneic rejection in the host to which the cells are administered.

[0155] Allogeneic cells are modified by using specific immunomodulatory factors introduced into cells or cell colonies. The modified cells escape immune rejection by regulating many different local immune pathways at the same time. Regardless of the genetic background, such genetically engineered cells can be "ready-made" to be transplanted into many receptors and are not rejected by the receptor immune system. This immunomodulatory method overcomes the requirement that systemic immune suppression (which may be dangerous to the recipient) is carried out in the transplant recipient. Therefore, although immunosuppressants can be applied to patients receiving modified cells described herein, the therapy need not include the application of immunosuppressants. This immunomodulatory method also overcomes expensive and impractical methods, obtains patient-specific iPS cells, manipulates regulatory cells, or induces chimerism by hematopoietic cell transplantation (HCT).

[0156] Cells can be genetically modified to express a group of transgenics, which are encoded as cytoplasmic, membrane-bound or locally acting and whose function is to alleviate immune function (e.g., leukocytes and NK cell activation of attacking transplants) or act as a gene product of a defense matrix for immune response (e.g., attacking leukocytes). The group of transgenics can be selected from genes that have an effect in the above-mentioned immunomodulatory pathways. Such genes include but are not limited to those provided in Table 1.

[0157] Table 1: Genes expressed by allogeneic cells that can be used for local immune suppression

[0158]

[0159] CC motif chemokine ligand 21 (Ccl21) is expressed by local lymph nodes where it serves to attract activated antigen presenting cells (APCs). This critical function provides an opportunity to "reverse" APC migration by overexpressing this gene on transplanted cells. Indeed, some melanomas express Ccl21 and recruit CCR7 + cells, which in turn can reorganize parts of their tumor stroma as "self". This leads to the directing of Cd4 + Recruitment of Tregs and maintenance of matrix reconstitution (Zindl et al., Science. 328: 697-8 (2010)). In fact, expression of Ccl21 on tumors can protect co-implanted Ccl21-deficient tumor cells from rejection in a syngeneic allograft setting (Shields et al., Science. 328: 749-52 (2010)). Ccl21b is the mouse ortholog of human Ccl21.

[0160] The amino acid sequences of mouse and human Ccl21 are:

[0161] Mouse Ccl21

[0162] MAQMMTLSLLSLVLALCIPWTQGSDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPEEGWVQNLMRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG(SEQ ID NO:1)

[0163] Human Ccl21

[0164] MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPA

[0165] KVVRSYRKQEPSLGCSIP

[0166] AILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGP(SEQ ID NO:2)

[0167] Expression of Cd47 in cord blood can promote the development of low-responsive T cells (Avice et al., J Immunol. 167: 2459-68 (2001)). Due to their lack of "self" presentation, erythrocytes also upregulate Cd47 to avoid dendritic cell activation (vandenBerg et al., Immunity. 43: 622-4 (2015)). Recently, it was shown that expression of human Cd47 increased engraftment in a mouse model of pig-to-human hematopoietic cell transplantation (Tena et al., Am J Transplant. 14: 2713-22 (2014)).

[0168] The amino acid sequences of mouse and human Cd47 are:

[0169] Mouse Cd47

[0170] MWPLAAALLLGSCCCGSAQLLFSNVNSIEFTSCNETVVIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTTTDQNFTSAKISV

[0171] SDLINGIASLKMDKRDAMVGNYTCEVTELSRE

[0172] GKTVIELKNRTVSWFSPNEKILIVIFPILAILLFWGKFGILTLKYKSSHTNKRIILLLVAGLVLTVIVVVGAILLIPGEKPVKNASGLGLIVISTGILILLQYNVFMTAFGMTSFTIAILITQVLGYVLALVGLCLCIMACEPVHGPLLISGLGIIALAELLGLVYMKFVASNQRTIQPPRNR(SEQ ID NO:3)

[0173] Human CD47

[0174] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIK TLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGGLLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE(SEQ ID NO:4)

[0175] Cd200 is also an important immune regulatory molecule; increased expression can reduce the severity of allograft rejection, autoimmunity and allergic diseases (Gorczynski et al., J Immunol. 172: 7744-9 (2004)). It has been shown that in vitro, APC expression of Cd200 suppresses the production of interferon gamma (IFN-γ) and cytolytic granules by activated Cd8+T cells (Misstear et al., J Virol. 86: 6246-57 (2012)). Most interestingly, overexpression of Cd200 increases the survival rate of skin and cardiac allografts in mice by promoting Foxp3+Treg cells (Gorczynski et al., Transplantation. 98: 1271-8 (2014)).

[0176] The amino acid sequences of mouse and human Cd200 are:

[0177] Mouse Cd200

[0178] MGSLVFRRPFCHLSTYSLIWGMAAVALSTAQVEVVTQDERKALHTTASLRCSLKTSQEPLIVTWQKKKAVSPENMVTYSKTHGVVIQPAYKDRINVTELGLWNSSITFWNTTLEDEGCYMCLFNTFGSQKVSGTACLTLYV QPIVHLHYNYFEDHLNITCSATARPAPAISWKGTGTGIENSTESHFHSNGTTSVTSILRVKDPKTQVGKEVICQVLYLGNVIDYKQSLDKGFWFSVPLLLSIVSLVILLVLISILLYWKRHRNQERGESSQGMQRMK(SEQ ID NO:5)

[0179] PeopleCd200

[0180] MERLVIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYV QPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDRGELSQGVQKMT(SEQ ID NO:6)

[0181] Spi6 is an endogenous inhibitor of the cytotoxic effector molecule granzyme B released by activated CD8+ T cells (Sun et al., J Biol Chem. 272: 15434-41 (1997)). Some data show that mesenchymal stem cells (MSCs) escape immune rejection by upregulating this molecule (El Haddad et al., Blood. 117: 1176-83 (2011)). It has also been recently demonstrated that the ability of dendritic cells to present antigens to cytotoxic T cells without being killed by contact-mediated cytotoxicity is mediated by Spi6 (Lovo et al., J Immunol. 188: 1057-63 (2012)). Spi6 is also known as Serpin B9.

[0182] The amino acid sequences of mouse Spi6 and its human counterpart Serpin B9 are:

[0183] Mouse Spi6

[0184] MNTLSEGNGTFAIHLLKMLCQSNPSKNVCYSPASISSALAMV

[0185] LLGAKGQTAVQISQALGL

[0186] NKEEGIHQGFQLLLRKLNKPDRKYSLRVANRLFADKTCEVLQTFKESSLHFYDSEMEQLSFAEEAEVSRQHINTWVSKQTEGKIPELLSGGSVDSETRLVLINALYFKGKWHQPFNKEYTMDMPFKINKDEKRPVQMMCREDTYNLAYVKEVQAQVLVMPYEGMELSLVVLLPDEGVDLSKVENNLTFEKLTAWMEADFMKSTDVEVFLPKFKLQEDYDMESLFQRLGVVDVFQEDKADLSGMSPERNLCVSKFVHQSVVEINEEGTEAAAASAIIEFCCASSVPTFCADHPFLFFIRHNKANSILFCGRFSSP(SEQ ID NO:7)

[0187] Human Serpin B9

[0188] METLSNASGTFAIRLLKILCQDNPSHNVFCSPVSISSALAMVL

[0189] LGAKGNTATQMAQALSL

[0190] NTEEDIHRAFQSLLTEVNKAGTQYLLRTANRLFGEKTCQFLS

[0191] TFKESCLQFYHAELKELS

[0192] FIRAAEESRKHINTWVSKKTEGKIEELLPGSSIDAETRLVLVNAIYFKGKWNEPFDETYTREMPFKINQEEQRPVQMMYQEATFKLA

[0193] HVGEVRAQLLELPYARKELSLLVLLPDDGVELS

[0194] TVEKSLTFEKLTAWTKPDCMKSTEVEVLLPKFKLQEDYDMESVLRHLGIVDAFQQGKADLSAMSAERDLCLSKFVHKSFVEVNEEGTEAAAASSCFVVAECCMESGPRFCADHPFLFFIRHNRANSILFCGRFSSP(SEQ IDNO:8)

[0195] Activated, cytotoxic Cd8+ can kill target cells by the expression of FasL, which binds to the FAS receptor and activates caspase-mediated apoptosis of targeted cells. However, many tumors develop "counterattacks" by raising FasL on their surfaces (Chen et al., JImmunol.171:1183-91 (2003)). The selective expression of FasL in the vasculature of human and mouse solid tumors has been associated with rare Cd8+T cell infiltration and a large number of FoxP3+Treg cells (Motz et al. Nat Med.20:607-15 (2014)). Recently, it has been shown that B lymphocytes also use the expression of FasL to kill T helper cells in the effector phase of immune response (Lundy et al., FrontImmunol.6:122 (2015)). FasL is a mouse ortholog of human FASLG.

[0196] The amino acid sequences of mouse FasL and its human counterpart, FASLG, are:

[0197] Mouse FasL

[0198] MQQPMNYPCPQIFWVDSSATSSWTPPGSVFPCPSSGPRGPDQRRPPPPPPPVSPLPPPSQPLPLPPLTPLKKKDHNTNLWLPVVFFMVLVALVGMGLGMYQLFHLQKELAELREFTNQSLKVSSFEKQIANPSTPSEKKEL RSVAHLTGNPHSRSIPLEWEDTYGTALISGVKYKKGSLVINEAGLYFVYSKVYFRGQSCNNQPLNHKVYMRNSKYPGDLVLMEEKRLNYCTTGQIWAHSSYLGAVFNLTSADHLYVNISQLSLINFEESKTFFGLYKL(SEQ ID NO:9)

[0199] FASLG

[0200] MQQPFNYPYPQIYWVDSSASSPWAPPGTVLPCPTSVPRRPGQRRPPPPPPPLPPPPPPPPLPPLPLPKKRGNHSTGLCLLVMFFMVLVALVGLGLGMFQLFHLQKELAELRESTSQMHTASSLEKQIGHPSPPPEKKE LRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL(SEQ ID NO:10)

[0201] PD-L1 is a key immunomodulatory molecule that binds to programmed cell death 1 (PD-1). PD-1 is expressed on T cells, and binding to PD-L1 results in T cell anergy (MacDonald et al., J Immunol. 126: 1671-5 (1981)).

[0202] The amino acid sequences of mouse and human PD-L1 are:

[0203] Mouse PD-L1

[0204] MRIFAGIIFTACCHLLRAFTITAPKDLYVVEYGSNVTMECRFP

[0205] VERELDLLALVVYWEKE

[0206] DEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGK RSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET(SEQ ID NO:11)

[0207] Human PDL1 (CD274)

[0208] MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPV TSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ IDNO:12)

[0209] Like those induced by allografts, the inflammatory environment attracts macrophages and inflammatory monocytes as well as many other innate immune cells. Milk fat globule epidermal growth factor-8 (Mfge-8) is expressed in many murine tumors (Neutzner et al., Cancer Res. 67:6777-85 (2007)) and has been shown to contribute to local immune suppression by polarizing incoming monocytes into suppressive M2-like macrophages (Soki et al., J Biol Chem. 289:24560-72 (2014)).

[0210] The amino acid sequences of mouse and human MFGE-8 are:

[0211] Mouse MFGE8

[0212] MQVSRVLAALCGMLLCASGLFAASGDFCDSSLCLNGGTCLTGQDNDIYCLCPEGFTGLVCNETERGPCSPNPCYNDAKCLVTLDTQRGDIFTEYICQCPVGYSGIHCETETNYYNLDGEYMFTTAVPNTAVPTPAPTPDLSNNLASRCSTQLGMEGGAIADSQISASSVYMGFMGLQRWGPELARLYRTGIVNAWTASNYDSKPWIQVNLLRKMRVSGVMTQGASRAGRAEYLKTFKVAYSLDGRKFEFIQDESGGDKEFLGNLDNNSLKVNMFNPTLEAQYIKLYPVSCHRGCTLRFELLGCELHGCSEPLGLKNNTIPDSQMSASSSYKTWNLRAFGWYPHLGRLDNQGKINAWTAQSNSAKEWLQVDLGTQRQVTGIITQGARDFGHIQYVASYKVAHSDDGVQWTVYEEQGSSKVFQGNLDNNSHKKNIFEKPFMARYVRVLPVSWHNRITLRLELLGC(SEQ ID NO:13)

[0213] Human MFGE8

[0214] MPRPRLLAALCGALLCAPSLLVALDICSKNPCHNGGLCEEISQEVRGDVFPSYTCTCLKGYAGNHCETKCVEPLGMENGNIANSQIAASSVRVTFLGLQHWVPELARLNRAGMVNAWTPSSNDDNPWIQVNLLRRMWVTGVVTQGASRLASHEYLKAFKVAYSLNGHEFDFIHDVNKKHKEFVGNWNKNAVHVNLFETPVEAQYVRLYPTSCHTACTLRFELLGCELNGCANPLGLKNNSIPDKQITASSSYKTWGLHLFSWNPSYARLDKQGNFNAWVAGSYGNDQWLQVDLGSSKEVTGIITQGARNFGSVQFVASYKVAYSNDSANWTEYQDPRTGSSKIFPGNWDNHSHKKNLFETPILARYVRILPVAWHNRIALRLELLGC(SEQ ID NO:14)

[0215] The potent killing potential of NK cells is also crucial in transplant rejection. NK cells can kill target cells lacking MHC class I molecules, as well as other cells in an inflammatory environment. H2-M3, a mouse homolog of human HLA-G, has recently been shown to have a regulatory effect on NK cells, allowing them to ignore cells lacking "self molecules" (Andrews et al., Nat Immunol. 13: 1171-7 (2012)). This is thought to be achieved by combining HLA-G, an immunosuppressive receptor on NK cells and T cells (Carosella et al., Adv Immunol. 127: 33-144 (2015)). H2-M3 is a mouse ortholog of human HLA-G.

[0216] The amino acid sequences of mouse H2-M3 and its human counterpart HLA-G are:

[0217] Mouse H2-M3

[0218] SIEEIPRMAPWMEKERPEYWKELKLKVKNIAQSARANLRTLLRYYNQSEGGSHILQWMVSCEVGPDMRLLGAHYQAAYDGSDYITLNEDLSSWTAVDMVSQITKSRLESAGTAEYFRAYVEGE CLELLHRFLRNGKEILQRADPPKAHVAHHPRPKGDVTLRCWALGFYPADITLTWQKDEEDLTQDMELVETRPSGDGTFQKWAAVVVPSGEEQRYTCYVHHEGLTEPLALKWGRSSQSSVVIMV(SEQ ID NO:15) Human HLA-G

[0219] MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKC EAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD(SEQ ID NO:16)

[0220] A group of transgenes including one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6A can be expressed in a cell. The cell can be, for example, a stem cell or a cell suitable for genome editing, such as a cell that can be used for treatment and / or differentiation into a therapeutic cell type. The stem cell can be, for example, an embryonic stem (ES) cell or an induced pluripotent stem (iPS) cell. The group of transgenes can include 1, 2, 3, 4, 5, 6, 7, or all 8 of these genes, or can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 of these genes. The cells may be further genetically modified to express one or more of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and / or IFNγR1 d39. The TGF-β transgene may be modified using methods known to those skilled in the art so as to express the gene product in a membrane-bound form (i.e., so that the gene product is expressed on the cell surface of the allograft). For example, a method for localizing TGF-β to the membrane is to co-express TGF-β with an additional transgene encoding the LRRC32 protein or any other polypeptide that causes TGF-β to be localized to the cell membrane. This protein anchors TGF-β to the membrane. (Tran DQ et al., Proc Natl Acad Sci USA 106: 13445-50 (2009)).

[0221] The amino acid sequence of IFNγR1 d39 is:

[0222] MGPQAAAGRMILLVVLMLSAKVGSGALTSTEDPEPPSVPVPTNVLIKSYNLNPVVCWEYQNMSQTPIFTVQVKVYSGSWTDSCTNISDHCCNIYGQIMYPDVSAWARVKA KVGQKESDYARSKEFLMCLKGKVGPPGLEIRRKKEEQLSVLVFHPEVVVNGESQGTMFGDGSTCYTFDYTVYVEHNRSGEILHTKHTVEKEECNETLCELNISVSTLDSRY CISVDGISSFWQVRTEKSKDVCIPPFHDDRKDSIWILVVAPLTVFTVVILVFAYWYTKKNSFKRKSIMLPKSLLSVVKSATLETKPESKYSLVTPHQPAVLESETVICEEP LSTVTAPDSPEAAEQEELSKETKALEEAGGSTSAMTPDSPPTPTQRRSSFSLLSSNQSGPCSLTAYHSRNGSDSGLVGSGSSISDLESLPNNNSETKMAEHDPPPVRKA(SEQ IDNO:17)

[0223] These genes can be human genes or mouse genes. In one embodiment, the gene has the same species as the recipient of the allograft recipient in which the cells are to be transplanted. In one embodiment, the gene has any species in which the function of the gene is conservative or the biological preparation designed has the agonist function of the endogenous counterpart. The method for introducing and expressing these transgenics in cells is described herein and is also known to those skilled in the art. The cells expressing these transgenics can be referred to as "masked" because they can escape allogeneic rejection without systemic immunosuppression and without the need for immunosuppressive drugs.

[0224] It is contemplated herein that cell populations derived from the above-described masked cells may also be used to generate local immune suppression when transplanted at a transplant site in an allogeneic recipient.

[0225] Before or after producing the masked cells of the present disclosure, the cells may first be modified to fail-safe cells. Fail-safe cells use cell division loci (CDLs) to control cell proliferation in animal cells. As provided herein, CDLs are loci whose transcription products are expressed during cell division. As described herein, CDLs may be genetically modified to include negative selective markers and / or gene expression systems based on inducible activators, which allow users to allow, eliminate and / or inhibit the proliferation of genetically modified cells by adding or removing appropriate inducers. Methods for preparing and using fail-safe cells are, for example, described in WO 2016 / 141480, the entire teachings of which are incorporated herein by reference. Cells may first be made fail-safe, and then masked. Alternatively, cells may first be masked, and then made fail-safe.

[0226] The cell can be a vertebrate cell, such as a mammalian cell, such as a human cell or a mouse cell. The cell can also be a vertebrate stem cell, such as a mammalian stem cell, such as a human stem cell or a mouse stem cell. Preferably, the cell or stem cell is suitable for genetic modification. Preferably, the user considers the cell or stem cell to have therapeutic value, meaning that the cell or stem cell can be used to treat a disease, disorder, defect or injury in a subject in need of treatment of the disease, disorder, defect or injury.

[0227] In some embodiments, the cell is a stem cell or progenitor cell (e.g., iPSC, embryonic stem cell, hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem or progenitor cell, germline stem cell, lung stem cell or progenitor cell, mammary stem cell, olfactory adult stem cell, hair follicle stem cell, pluripotent stem cell, amniotic membrane stem cell, cord blood stem cell or neural stem cell or progenitor cell). In some embodiments, the stem cell is an adult stem cell (e.g., somatic stem cell or tissue-specific stem cell). In some embodiments, the stem cell or progenitor cell is capable of differentiation (e.g., stem cell is omnipotent, multipotent or multipotent). In some embodiments, the cell is separated from embryonic or neonatal tissue. In some embodiments, the cell is a fibroblast, a monocyte precursor, a B cell, an exocrine cell, a pancreatic progenitor cell, an endocrine progenitor cell, a hepatoblast, a myoblast, a preadipocyte, a progenitor cell, a hepatocyte, a chondrocyte, a smooth muscle cell, a K562 human erythroleukemia cell line, a bone cell, a synoviocyte, a tendon cell, a ligament cell, a meniscus cell, a fat cell, a dendritic cell or a natural killer cell. In some embodiments, the cell is manipulated (e.g., transformed or differentiated) into a myoblast, an erythrocyte-megakaryocyte, an eosinophil, an iPS cell, a macrophage, a T cell, a pancreatic beta cell, a neuron, a cardiomyocyte, a blood cell, an endocrine progenitor cell, an exocrine progenitor cell, a duct cell, acinar cell, an alpha cell, a beta cell, a delta cell, a PP cell, a hepatocyte, a bile duct cell or a brown adipocyte. In some embodiments, the cell is a muscle cell (e.g., skeletal muscle, smooth muscle, or cardiomyocyte), an erythroid-megakaryocyte, an eosinophil, an iPS cell, a macrophage, a T cell, a pancreatic beta cell, a neuron, a cardiomyocyte, a blood cell (e.g., a red blood cell, a white blood cell, or a platelet), an endocrine progenitor cell, an exocrine progenitor cell, a duct cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a PP cell, a hepatocyte, a bile duct cell, or a white or brown adipocyte.In some embodiments, the cell is a hormone-secreting cell (e.g., a cell that secretes insulin, oxytocin, endorphin, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormone, bombesin, cholecystokinin, testosterone, estrogen or progesterone, renin, ghrelin, amylin or pancreatic polypeptide), an epidermal keratinocyte, an epithelial cell (e.g., an exocrine gland secretory epithelial cell, a thyroid epithelial cell, a keratinized epithelial cell, a gallbladder epithelial cell, or a surface epithelial cell of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, or vagina), a kidney cell, a skeletal joint synovial cell, a periosteal cell, a bone cell (e.g., an osteoclast or osteoblast), a perichondrial cell (e.g., a chondroblast or chondrocyte), a cartilage cell (e.g., a In some embodiments, the cell is a somatic cell. In some embodiments, the cell is derived from the skin or other organs, such as the heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach. The cell may be from a human or other mammal (e.g., a rodent, non-human primate, bovine or porcine cell). It is contemplated herein that the masked cells may be used in cell-based therapies where it may be desirable to escape allogeneic rejection at a localized transplant site.

[0228] In some embodiments, the cloaked cells described herein survive in a host without stimulating a host immune response for a week or longer (e.g., a week, two weeks, a month, two months, three months, six months, a year, two years, three years, four years, five years, or longer, such as for the life of the cell and / or its progeny). The cells maintain their expression as long as the cloaking transgene survives in the host (e.g., if the cloaking transgene is no longer expressed, the cloaked cells can be removed by the host's immune system). In some embodiments, the cloaked cells further express a transgene that encodes a protein that allows detection of the cloaked cells in vivo (e.g., a fluorescent protein such as GFP or other detectable marker).

[0229] It is contemplated herein that a combination of cloaked cells and fail-safe cells may be used in cell-based therapies where it may be desirable to evade allogeneic rejection at a localized transplant site while also being able to eliminate cells that exhibit an undesirable growth rate, whether such cells are generated prior to or after transplantation of the cells into the host. The combined cloaking and fail-safe techniques allow for localized immune protection while addressing the risk that the recipient will develop a malignancy due to the localized immune containment being provided by the cells.

[0230] Methods for producing masked cells

[0231] The compositions and methods described herein can be used to mitigate rejection of allogeneic cells by masking the expression of transgenes.A variety of methods have been established for delivering proteins to mammalian cells and for stably expressing protein-encoding genes in mammalian cells, which methods can be used to generate the masked cells described herein.

[0232] Polynucleotides encoding masked proteins or therapeutic agents

[0233] One platform that can be used to achieve therapeutically effective expression of masked proteins or therapeutic agents in mammalian cells is via stable expression of genes encoding masked proteins or therapeutic agents (e.g., by integration into the nuclear or mitochondrial genome of mammalian cells, or by free concatemer formation in the nucleus of mammalian cells). The gene is a polynucleotide encoding the primary amino acid sequence of the corresponding protein. In order to introduce exogenous genes into mammalian cells, the gene can be incorporated into a vector. The vector can be introduced into the cell by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and by encapsulating the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail in, for example, Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014); and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

[0234] Masking proteins or therapeutic agents can also be introduced into mammalian cells by targeting a vector containing a portion of a gene encoding the masking protein or therapeutic agent to cell membrane phospholipids. For example, a vector can be targeted to phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to the VSV-G protein, a viral protein that has an affinity for all cell membrane phospholipids. Such constructs can be generated using methods well known to those skilled in the art.

[0235] Recognition and binding of a polynucleotide encoding a masking protein or therapeutic agent by a mammalian RNA polymerase is important for gene expression. Thus, sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and promote assembly of transcription complexes at transcription start sites may be included in the polynucleotide. Such sequence elements include, for example, mammalian promoters, whose sequences can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerases.

[0236] Polynucleotides suitable for the compositions and methods described herein also include those polynucleotides encoding masking proteins or therapeutic agents downstream of mammalian promoters. Promoters that can be used to express masking proteins or therapeutic agents in mammalian cells include constitutive promoters. Constitutive promoters include CAG promoters, cytomegalovirus (CMV) promoters, EF1α promoters, and PGK promoters. Alternatively, promoters derived from viral genomes can also be used to stably express these agents in mammalian cells. Examples of functional viral promoters that can be used to promote mammalian expression of these agents include adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, tk promoters of HSV, mouse mammary tumor virus (MMTV) promoters, LTR promoters of HIV, promoters of Moloney viruses, Epstein Barr virus (EBV) promoters, and Rous sarcoma virus (RSV) promoters.

[0237] Once the polynucleotides encoding masking proteins or therapeutic agents described herein below are incorporated into the nuclear DNA of mammalian cells, the transcription of such polynucleotides can be induced by methods known in the art. For example, expression can be induced by exposing mammalian cells to external chemical agents (such as agents that regulate the binding of transcription factors and / or RNA polymerases to mammalian promoters and thus regulate gene expression). Chemical agents can be used to promote the binding of RNA polymerases and / or transcription factors to mammalian promoters, for example, by removing the repressor proteins bound to the promoter. Alternatively, chemical agents can be used to enhance the affinity of mammalian promoters to RNA polymerases and / or transcription factors, so that the transcription rate of genes located downstream of the promoter is increased in the presence of chemical agents. Examples of chemical agents that enhance polynucleotide transcription by the above-mentioned mechanism include tetracycline and doxycycline. These agents are commercially available (Life Technologies, Carlsbad, CA), and can be applied to mammalian cells to promote gene expression according to established protocols.

[0238] Other DNA sequence elements that may be included in the nucleic acid vectors used in the compositions and methods described herein include enhancer sequences. Enhancers represent another type of regulatory element, which induces conformational changes in the polynucleotide containing the target gene so that the DNA adopts a three-dimensional orientation that is conducive to the combination of transcription factors and RNA polymerases at the transcription start site. Therefore, the polynucleotides used in the compositions and methods described herein include those that encode masking proteins or therapeutic agents and additionally include mammalian enhancer sequences. Many enhancer sequences are now known from mammalian genes, and examples include enhancers from genes encoding mammalian globulins, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers used in the compositions and methods described herein also include enhancers derived from viral genetic material that can infect eukaryotic cells. Examples include SV40 enhancers on the rear side of the replication origin (bp 100-270), cytomegalovirus early promoter enhancers, polyoma enhancers on the rear side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of transcription of eukaryotic genes are disclosed in Yaniv et al., Nature 297:17 (1982). The enhancer can be spliced ​​into a vector containing a polynucleotide encoding a masking protein or therapeutic agent, for example, at a position 5' or 3' to the gene. In a preferred orientation, the enhancer is located 5' to the promoter, which in turn is located 5' relative to the polynucleotide encoding the masking protein or therapeutic agent.

[0239] The nucleic acid vectors described herein may include a woodchuck post-transcriptional regulatory element (WPRE). The WPRE acts at the transcriptional level by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of nascent transcripts, thereby increasing the total amount of mRNA in the cell. Adding the WPRE to the vector can result in significant improvements in the level of transgene expression from several different promoters in vitro and in vivo.

[0240] In some embodiments, the nucleic acid vector used in the compositions and methods described herein comprises a reporter gene sequence, which may be applicable to verifying gene expression in, for example, specific cells and tissues. The reporter gene sequence that may be provided in the transgenic includes a DNA sequence encoding beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and other enzymes known in the art. When associated with the regulatory element driving its expression, the reporter gene sequence provides a signal that can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescent or other spectrometric determinations, fluorescence activated cell sorting determinations and immunoassays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example, in the case where the marker sequence is a LacZ gene, the presence of a carrier carrying a signal is detected by a determination for beta-galactosidase activity. When the transgenic is green fluorescent protein or luciferase, the carrier carrying a signal can be visually measured by color or luminescence in a luminometer.

[0241] Techniques used to introduce transgenes into cells

[0242] Transfection

[0243] Techniques that can be used to introduce transgenes, such as masked transgenes or therapeutic transgenes as described herein, into target cells (e.g., mammalian cells) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the target cells. Mammalian cells (e.g., human cells) subjected to an external electric field in this manner are then susceptible to taking up exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. Similar techniques Nucleofection TM The use of an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. TM Protocols that can be used to perform this technique are described in detail in, for example, Distler et al., Experimental Dermatology 14:315 (2005) and US 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0244] Other techniques that can be used to transfect target cells include extrusion perforation methods. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to applied pressure. The advantage of this technique is that no vector is required for delivering nucleic acids to cells, such as human target cells. Extrusion perforation is described in detail in, for example, Sharei et al., Journal of Visualized Experiments 81: e50980 (2013), the disclosure of which is incorporated herein by reference.

[0245] Lipofection represents another technique that can be used to transfect target cells. This method involves loading nucleic acids into liposomes, which typically present cationic functional groups, such as quaternary amines or protonated amines, to the outside of the liposomes. Due to the anionic nature of the cell membrane, this promotes electrostatic interactions between liposomes and cells, which ultimately leads to the uptake of exogenous nucleic acids, for example, by direct fusion of liposomes with cell membranes or endocytosis of complexes. Lipofection is described in detail, for example, in U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that utilize ionic interactions with cell membranes to stimulate the uptake of exogenous nucleic acids include contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge that facilitates interaction with cell membranes include activated dendrimers (described, for example, in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this method utilizes an applied magnetic field to direct the uptake of nucleic acids. This technology is described in detail, for example, in US 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0246] Another useful tool for inducing target cells to take up exogenous nucleic acids is laser transfection (also known as optical transfection), which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to, and in some cases superior to, electroporation.

[0247] Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials, such as carbon nanofibers, carbon nanotubes and nanowires. Needle-shaped nanostructures are synthesized perpendicular to the surface of the substrate. DNA containing genes intended for intracellular delivery is attached to the nanostructure surface. The chip with an array of these needles is then pressed against cells or tissues. Cells pierced by the nanostructures can express the delivered genes. Examples of this technology are described in Shalek et al., PNAS 107: 1870 (2010), the disclosure of which is incorporated herein by reference.

[0248] Magnetorfection can also be used to deliver nucleic acids to target cells. The principle of magnetorfection is to associate nucleic acids with cationic magnetic nanoparticles. Magnetic nanoparticles are made of completely biodegradable iron oxide and coated with specific cationic proprietary molecules that vary according to the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved through salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells under the influence of the external magnetic field generated by the magnet. This technology is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0249] Another useful tool for inducing target cells to take up exogenous nucleic acids is sonoporation, which is a technique involving the use of sound (usually ultrasonic frequencies) to change the permeability of the cytoplasmic membrane, permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail in, for example, Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0250] Microvesicles represent another potential medium that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of glycoprotein VSV-G and, for example, genome modification proteins (such as nucleases) can be used to effectively deliver proteins to cells, which then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of cells for covalent incorporation into target polynucleotides, such as genes or regulatory sequences. Such vesicles (also referred to as Gesicles) are used for the genetic modification of eukaryotic cells, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [Abstract]. In: Methylation changes in early embryonic genes in cancer [Abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; May 13, 2015, Abstract No. 122 is described in detail.

[0251] Viral infection

[0252] In addition to achieving high transcription and translation rates, stable expression of exogenous genes in mammalian cells can also be achieved by integrating polynucleotides containing the genes into the nuclear genome of mammalian cells. A variety of vectors have been developed for delivering and integrating polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of expression vectors are disclosed in, for example, WO 1994 / 011026 and are incorporated herein by reference. Expression vectors for compositions and methods described herein contain masking transgenes or therapeutic transgenes, as well as other sequence elements, for example, for expressing these agents and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express masking transgenes or therapeutic transgenes include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing masking transgenes or therapeutic transgenes contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct the efficient transcription of genes carried on the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0253] Viral vectors for nucleic acid delivery

[0254] The viral genome provides a rich source of vectors, which can be used to effectively deliver the target gene to the genome of the target cell (e.g., mammalian cells, such as human cells). The viral genome is a particularly useful vector for gene delivery, because the polynucleotides contained in such genomes are usually incorporated into the nuclear genome of mammalian cells by universal or specific transduction. These processes occur as part of the natural viral replication cycle, and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., Retroviridae virus vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses (including adenoviruses, herpes viruses (e.g., herpes simplex viruses type 1 and type 2, Epstein-Barr viruses, cytomegaloviruses), and poxviruses. (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). For example, other viruses include Norwalk virus, togavirus, flavivirus, reovirus, papillomavirus, hepadnavirus, human papillomavirus, human foamy virus, and hepatitis virus. Examples of retroviruses include: avian leukosis-sarcoma virus, avian C virus, mammalian C virus, B virus, D virus, oncorretrovirus, HTLV-BLV group, lentivirus, alpha retrovirus, gamma retrovirus, spumavirus (Coffin, JM, Retroviridae: The Viruses and their replication, Virology, 3rd edition (Lippincott-Raven, Philadelphia, 1996)). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it relates to viral vectors for gene therapy.

[0255] AAV vectors for nucleic acid delivery

[0256] In some embodiments, the masking transgene or therapeutic transgene described herein is incorporated into the rAAV vector and / or virion to facilitate their introduction into the cell. The rAAV vector that can be used for the compositions and methods described herein is a recombinant nucleic acid construct comprising (1) a promoter, (2) a heterologous sequence to be expressed (e.g., a masking transgene or therapeutic transgene described herein), and (3) a viral sequence that promotes the integration and expression of the heterologous gene. The viral sequence may include those sequences of the AAV required for cis-DNA replication and packaging (e.g., functional ITR) into virions. Such rAAV vectors may also contain markers or reporter genes. Useful rAAV vectors have one or more of the AAV WT genes that are completely or partially deleted, but retain functional flanking ITR sequences. The AAV ITRs may have any serotype suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0257] The transgenes and vectors described herein (e.g., promoters operably linked to masking transgenes or therapeutic transgenes) can be incorporated into rAAV virions to facilitate the introduction of polynucleotides or vectors into cells. The capsid protein of AAV constitutes the external non-nucleic acid portion of the virion and is encoded by the AAVcap gene. The cap gene encodes three viral coat proteins VP1, VP2, and VP3, which are required for the assembly of virions. The construction of rAAV virions has been described in, for example, US 5,173,414; US 5,139,941; US ​​5,863,541; US ​​5,869,305; US 6,057,152; and US 6,376,237; and Rabinowitz et al., J. Virol. 76: 791 (2002) and Bowles et al., J. Virol. 77: 423 (2003), the disclosures of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0258] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43, and rh74. The construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0259] Also used in combination with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype different from a given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). The construction and use of pseudotyped rAAV virions are known in the art and are described, for example, in Duan et al., J. Virol. 75: 7662 (2001); Halbert et al., J. Virol. 74: 1524 (2000); Zolotukhin et al., Methods, 28: 158 (2002); and Auricchio et al., Hum. Molec. Genet. 10: 3075 (2001).

[0260] AAV virions with mutations in the virion capsid can be used to infect specific cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations to help AAV target specific cell types. The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants are described in Wu et al., J. Virol. 74: 8635 (2000). Other rAAV virions that can be used in the methods described herein include those capsid hybrids produced by molecular breeding of viruses and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25: 436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19: 423 (2001).

[0261] Genome editing

[0262] In addition to the above, a variety of tools that can be used to incorporate target genes into target cells such as mammalian cells have been developed. A method that can be used to incorporate polynucleotides encoding target genes into target cells relates to the use of transposons. Transposons are polynucleotides that encode transposases and contain target polynucleotide sequences or genes that flank 5' and 3' excision sites. Once transposons have been delivered into cells, the expression of transposase genes begins and produces an active enzyme that cracks target genes from transposons. This activity is mediated by the site-specific recognition of transposase excision sites of transposons. In some cases, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from transposons, target genes can be integrated into the genome of mammalian cells by similar excision sites present in the nuclear genome of cells through transposase catalytic cracking. This allows the target gene to be inserted into the nuclear DNA of cracking at the complementary excision site, and the phosphodiester bond that covalently connects the target gene to the DNA of the mammalian cell genome is subsequently incorporated into the process. In some cases, the transposon can be a retrotransposon, so that the gene encoding the target gene is first transcribed into an RNA product and then reverse transcribed into DNA, which is then incorporated into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (e.g., described in detail in WO 2010 / 085699) and the sleeping beauty transposon (e.g., described in detail in US 2005 / 0112764), the disclosures of each of which are incorporated herein by reference as they relate to transposons for gene delivery to target cells.

[0263] Another tool for integrating the target gene into the genome of the target cell is the clustered regularly spaced short palindromic repeats (CRISPR) / Cas system, which is a system that originally evolved into an adaptive defense mechanism for viral infection in bacteria and archaea. The CRISPR / Cas system includes palindromic repeats and related Cas9 nucleases in plasmid DNA. This collection of DNA and protein guides the site-specific DNA cleavage of the target sequence by first incorporating foreign DNA into the CRISPR locus. The polynucleotides containing the repeat sequence-spacer elements of these foreign sequences and CRISPR loci are then transcribed in the host cell to produce guide RNAs, which can then anneal to the target sequence and locate the Cas9 nuclease to this site. In this way, highly site-specific cas9-mediated DNA cleavage can be produced in foreign polynucleotides because the interaction of cas9 in the vicinity of the target DNA molecule is controlled by RNA:DNA hybridization. As a result, the CRISPR / Cas system can be designed to cleave any target DNA molecule of interest. This technology has been used to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31: 227 (2013)) and can be used as an effective means for site-specific editing of the target cell genome so as to cleave DNA before incorporating the gene encoding the target gene. Using CRISPR / Cas to regulate gene expression has been described, for example, in U.S. Patent No. 8,697,359, the disclosure of which is incorporated herein by reference because it relates to genome editing using the CRISPR / Cas system. Before incorporating the target gene into the target cell, alternative methods for site-specific cleavage of genomic DNA include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain guide polynucleotides for locating to specific target sequences. Instead, target specificity is controlled by the DNA binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov et al., Nature Reviews Genetics 11:636 (2010); and Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosures of each of which are incorporated herein by reference as they relate to compositions and methods for genome editing.

[0264] Additional genome editing techniques that can be used to incorporate a polynucleotide encoding a target gene into the genome of a target cell include the use of ARCUS, which can be rationally designed to site-specifically cleave genomic DNA. TMMeganucleases. Given that a defined structure-activity relationship has been established for such enzymes, it is advantageous to use these enzymes to incorporate genes encoding target genes into the genome of mammalian cells. Single-chain meganucleases can be modified at certain amino acid positions to produce nucleases that selectively cleave DNA at the desired position, thereby enabling site-specific incorporation of the target gene into the nuclear DNA of the target cell. These single-chain nucleases have been extensively described in, for example, U.S. Patent Nos. 8,021,867 and 8,445,251, the disclosures of which are incorporated herein by reference as they relate to compositions and methods for genome editing.

[0265] Masking transgene expression

[0266] The masking transgenes described herein (e.g., one or any combination of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and SerpinB9 (Spi6)) are expressed in an amount sufficient to produce a masking effect (e.g., an amount sufficient to prevent rejection when injected into a subject, e.g., a mammalian subject, such as a mouse, rat, or human). If subcutaneous injection of masked cells produces a teratoma that is not cleared by the subject's immune system, then the transgene expression can be considered to produce a masking effect. The masking transgene is also expressed at a level sufficient to promote the production of the protein encoded by the transgene. Protein production can be detected using conventional methods known to those skilled in the art (e.g., immunohistochemistry, Western blot analysis, or other methods that allow visualization of proteins). Preferably, expression of the masking transgene is such that all eight proteins encoded by the masking transgene (PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6) are detectable in the masked cells (e.g., by immunohistochemistry using antibodies against proteins encoded by the masking transgene).

[0267] In some embodiments, the masked transgene is expressed in the masked cells at a level similar to the level of expression of the endogenous gene in activated leukocytes, such as T cells (e.g., activated leukocytes from the same species, such as activated leukocytes isolated from lymphoid organs, e.g., expression in masked mouse cells is similar to expression in activated leukocytes isolated from murine lymphoid organs). The expression of one or more masking transgenes (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)) is greater than or equal to the expression of endogenous genes in activated leukocytes (e.g., T cells) from the same species (e.g., the expression level of the masking transgene is equal to the expression level of the endogenous gene in the activated leukocytes, or 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more higher than the expression level of the endogenous gene in the activated leukocytes). In some embodiments, all 8 masking transgenes are expressed at a level greater than or equal to the expression level of the endogenous gene in activated leukocytes from the same species. Activated leukocytes may be isolated from lymphoid organs, or leukocytes, such as T cells, may be activated in vitro using anti-CD3 / CD28 beads or other methods employed by those skilled in the art (see, e.g., Frauwith and Thompson, J. Clin Invest 109:295-299 (2002); and Trickett and Kwan, J Immunol Methods 275:251-255 (2003)). Transgene expression in masked cells may also be compared to gene expression levels reported in profiling studies of activated T cells (see, e.g., Palacios et al., PLOSone 2:el222 (2007)). In some embodiments, masked transgene expression is compared to expression of endogenous genes in a wild-type version of the cell (e.g., a stem cell, such as an embryonic stem cell from the same species as the masked cell). The expression of one or more masking transgenes (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)) is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1,000 times or more in the masked cells as compared to the expression of the endogenous gene in an unmodified wild-type cell of the same cell type as the masked cells (e.g., a stem cell, such as an embryonic stem cell from the same species).In some embodiments, all eight masked transgenes are expressed at levels greater than the expression level of the endogenous gene in a wild-type version of a cell (e.g., a stem cell, such as an embryonic stem cell from the same species as the masked cell) (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 times or more). Gene expression can be assessed by direct comparison to isolated ES cells or by comparison to stem cell expression (e.g., ES cell expression) in the Project Grandiose dataset (www.stemformatics.org / project_grandiose). Gene expression can be measured using techniques known in the art (e.g., quantitative polymerase chain reaction (qPCR)).

[0268] Methods for providing local immunosuppression at the transplant site

[0269] Also featured are methods of providing local immunosuppression at the transplant site.

[0270] The method comprises providing a cell; and expressing a set of transgenes in the cell, each transgene encoding a gene product that is cytoplasmic, membrane-bound or locally acting and whose function is to reduce the activation of leukocytes and NK cells that attack the transplant or serve as a defense mechanism against the attacking leukocytes.

[0271] The group of transgenes includes one or more (e.g., two, three, four, five, six, seven or all eight) of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and SerpinB9 (Spi6). In one embodiment, the group of transgenes includes Pd-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8 and Spi6.

[0272] Optionally, the method further comprises expressing one or more of the following transgenes in the cell: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39. In one embodiment, the TGF-β or biologic is locally acting.

[0273] Techniques for introducing various genetic modifications, such as transgenes, into animal cells are described herein and generally known in the art.

[0274] In one embodiment of the method, the cell is a stem cell, a cell suitable for genome editing, and / or a source of therapeutic cell types (e.g., cells that can be differentiated into lineage-restricted cells for cell therapy or cells of desired target tissues). In one embodiment, the cell is an embryonic stem cell, an induced pluripotent stem cell, an adult stem cell, a tissue-specific stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem cell or progenitor cell, a germline stem cell, a lung stem cell or progenitor cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, a pluripotent stem cell, an amniotic membrane stem cell, an umbilical cord blood stem cell, or a neural stem cell or progenitor cell. In some embodiments, the cell is derived from a target tissue, such as skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach. In some embodiments, the cell is a fibroblast, an epithelial cell or an endothelial cell. The cell can be a vertebrate cell, such as a mammalian cell, such as a human or mouse cell. In some embodiments, the cells modified to express one or more (e.g., two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are cells in a tissue or organ to be transplanted. In some embodiments, the cloaked cells (e.g., cloaked stem cells) are differentiated into tissues or organs for transplantation in vitro using methods known to those skilled in the art.

[0275] In some embodiments, one million to one hundred billion masked cells (e.g., 1×10 6 , 2x 10 6 , 3x10 6 , 4x 10 6 , 5x 10 6 , 6x 10 6 , 7x 10 6 , 8x 10 6 , 9x10 6 , 1x 10 7 , 2x 10 7 , 3x 10 7 , 4x 10 7 , 5x 10 7 , 6x 10 7 , 7x 10 7 , 8x10 7 , 9x 10 7 , 1x 10 8 , 2x 10 8 , 3x 10 8 , 4x 10 8, 5x 10 8 , 6x 10 8 , 7x10 8 , 8x10 8 , 9x 10 8 , 1x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 , 5x 10 9 , 6x10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , 1x 10 10 , 2x 10 10 , 3x 10 10 , 4x 10 10 , 5x 10 10 , 6x 10 10 , 7x 10 10 , 8x 10 10 , 9x 10 10 or 1x 10 11 The transplanted cells are administered to a subject at or near a transplantation site, or to an organ or tissue to be transplanted.

[0276] Techniques for transplanting genetically modified cells into an allogeneic host at a transplantation site are described herein and generally known in the art.

[0277] Expression of therapeutic agents by masked cells

[0278] The cells covered as described herein may be further modified to express therapeutic agents. In some embodiments, the therapeutic agent is a protein. The therapeutic protein may be a wild-type form of a protein lacking in a subject, such as a protein produced by a cell mutation of the subject or produced in insufficient amounts (e.g., produced at low levels or not produced). In some embodiments, the therapeutic protein is an inhibitory antibody (e.g., an antibody that blocks or neutralizes the function of a protein). The cells covered may be modified to produce inhibitory antibodies to treat a subject suffering from a disease or disorder related to excessive or abnormal production of a protein (e.g., produced by cells that do not normally produce proteins, producing the protein at a time or location that does not normally produce proteins, or producing an excessive amount of protein) or a subject at risk of developing the disease or disorder. In some embodiments, the therapeutic antibody is an agonist antibody (e.g., an activating antibody). The agonist antibody may act by binding to and activating an endogenous receptor (e.g., inducing or increasing signal transduction downstream of receptor activation or changing the conformation of an endogenous receptor to an open or active state). The masked cells can be modified to produce agonist antibodies to treat subjects suffering from or at risk of developing a disease or disorder associated with activation of a receptor or signaling pathway. The masked cells can be modified to produce therapeutic proteins or antibodies using the methods described herein or using other methods known to those skilled in the art. Masked cells that produce secreted proteins or antibodies can be delivered as circulating cells, injected into tissues, organs or body parts that require therapeutic proteins or antibodies, or injected subcutaneously to produce masked subcutaneous tissue. Masked cells that produce transmembrane or membrane-bound proteins can be injected at or near the site of endogenous cells that respond to the therapeutic protein.

[0279] In some embodiments, the masked cells described herein provide a wild-type copy of a gene that is mutated in a subject (e.g., a masked cell is a "wild-type cell" that does not have the genetic cause of the disease and expresses one, two, three, four, five, six, seven, or all eight of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and SerpinB9 (Spi6). Such cells can be used to treat subjects with a disease or condition caused by a mutation in an endogenous gene (e.g., a subject with a metabolic disorder associated with one or more mutations described below).

[0280] A list of exemplary therapeutic agents that can be administered with or produced by the cloaked cells and related diseases or conditions that can be treated using these therapeutic agents is provided in Table 2 below.

[0281] Table 2: Exemplary therapeutic agents that can be administered with or expressed by cloaked cells to treat disease

[0282]

[0283]

[0284]

[0285]

[0286] Inducible systems for expression of therapeutic agents

[0287] If it is necessary to continuously administer a therapeutic agent expressed by the masked cells to treat a disease or illness, a constitutive promoter described herein or known to those skilled in the art (e.g., CAG, CMV or other constitutive promoters) can be used to express the therapeutic agent. If a therapeutic agent is needed intermittently (e.g., a therapeutic agent is needed during a relapse or attack that occurs during a disease or illness, but not required when the subject is asymptomatic), the therapeutic agent can be expressed by an inducible promoter, which provides the ability to express the therapeutic agent only when needed. An exemplary class of therapeutic agents that can be delivered using an inducible promoter is a TNFα inhibitor. TNFα inhibitors are currently used to treat rheumatoid arthritis, but are only administered intermittently during an attack of joint inflammation because the constitutive administration of TNFα can lead to systemic immunosuppression. If the masked cells are modified to express TNFα inhibitors under the control of an inducible promoter, the masked cells can be used to deliver TNFα intermittently, so there is no need for repeated injections. Other therapeutic agents with potential adverse effects if administered continuously can also be expressed intermittently using an inducible promoter as described herein. Exemplary inducible expression systems are described below.

[0288] Tetracycline response element

[0289] A widely used inducible expression system is based on tetracycline-controlled transcriptional activation. In this system, the antibiotic tetracycline or one of its derivatives (e.g., doxycycline) is used to reversibly activate or inhibit gene expression. To use this system, a tetracycline response element (TRE) is placed upstream of the target gene (e.g., a therapeutic transgene to be expressed by the shielded cells), usually together with a minimal promoter with very low basal expression. A protein called rtTA (which also needs to be expressed by the shielded cells) binds to the TRE and activates transcription in the presence of tetracycline or doxycycline. When tetracycline or doxycycline is removed, rtTA no longer binds to the TRE, and the target gene is no longer expressed. An advanced version of this system, the Tet-On Advanced transactivator (rtTA2 s -M2) and Tet-On 3G may be particularly useful for human therapy because they are human codon-optimized and respond to low concentrations of doxycycline.

[0290] Light-induced system

[0291] Another method for inducible activation of gene expression involves the use of optogenetics, which uses light-sensitive proteins to manipulate gene expression. The latest developments in optogenetics that can be used to inducibly express therapeutic agents in veiled cells involve a class of proteins that undergo conformational changes and dimerize in response to blue light. These proteins have been fused to DNA binding and transcription components that have been shown to bind to specific promoter sequences and activate transcription when brought together by exposure to blue light (Wang et al., Nat Methods, 9: 266-269, 2012). This method of inducibly activating gene expression can be used to control the production of therapeutic agents in subcutaneously administered veiled cells, because blue light can be irradiated onto the skin near the veiled subcutaneous tissue to induce the veiled cells to produce therapeutic agents.

[0292] Radiogenetics

[0293] The third method of inductively activating gene expression (e.g., expressing therapeutic agents by concealed cells) involves the use of radio waves. In one version of the radio wave inducible expression system, the TRPV1 receptor is fused to the GFP binding domain and co-expressed with a form of ferritin connected to GFP (Stanley et al., Nat Med 21:92-98, 2015). GFP-ferritin binds to the GFP binding domain of the TRPV1 receptor. When radio waves of a specific frequency are applied to cells, ferritin interacts with TRPV1 and allows the influx of calcium, which activates the transcription factor NFAT. If the therapeutic agent is operably connected to a NFAT-sensitive promoter element (such as SRE-CRE-NFATRE) and co-expressed with TRPV1-GFP and GFP-ferritin, this system can be used to inductively express the therapeutic agent. Radio wave-induced expression provides the advantage of being able to induce expression in cells away from the outside of the body because radio waves can pass through tissues. For example, radiogenetics can be used to regulate gene expression in the retina. Therefore, this method can be used to inducibly express therapeutic transgenes in masked cells via non-invasive and harmless radio waves.

[0294] Destabilizing domain system

[0295] Gene expression can also be regulated using a destabilizing domain system. A transgene encoding a target protein (e.g., a therapeutic agent described herein) may also include a destabilizing domain so that the resulting protein product includes a target protein fused to a destabilizing domain. Exemplary destabilizing domains include mutants of human FK506- and rapamycin binding protein (FKBP12), which confer instability to the protein fused thereto. FKBP12 mutants include N-terminal mutants F15S, V24A, H25R, E60G, and L106P, and C-terminal mutants M66T, R71G, D100G, D100N, E102G, and K105I, as characterized in Banaszynski et al., Cell 126:995 (2006), the disclosure of which is incorporated herein by reference as it relates to a FKBP12 destabilizing domain. Destabilizing domains promote protein degradation. When it is desired to express a target protein (e.g., a therapeutic agent), a small molecule synthetic ligand can be used to stabilize a protein containing a destabilizing domain. The small molecule ligand Shield-1 (Shld1) can be used to stabilize proteins containing FKBP12 mutants by protecting them from degradation. Other destabilizing domains that can be used to regulate the expression of target proteins include mutants of Escherichia coli dihydrofolate reductase (ecDHFR) and mutants of human estrogen receptor ligand binding domain (ERLBD), which impart instability when fused to the target protein, thereby causing degradation, and can be stabilized by the small molecule ligand trimethoprim (TMP) or by CMP8 or 4-hydroxytamoxifen (4OHT), respectively, as described in Iwamoto et al., Chem Biol. 17: 981 (2010) and Miyazaki et al., J Am Chem Soc., 134: 3942 (2012), the disclosures of which are incorporated herein by reference as they relate to destabilizing domain systems.

[0296] Coumarate switch inducible system

[0297] Another method for the inducible activation of gene expression relates to the use of coumarate gene switch system. In the repressor configuration of this system, regulation is mediated by the combination of repressor (CymR) and the operator site (CuO) placed downstream of the strong constitutive promoter. Adding coumarate (a small molecule) can alleviate repression, thereby allowing transgenic expression. Alternatively, reverse-coumarate-transactivator (rcTA) can be inserted into the upstream of the minimum CMV promoter, and the promoter can be operably connected to the transgenic encoding therapeutic agent. 6 times of repetitions (6xCuO) of the coumarate operon can be inserted just before the translation initiation (ATG) of the therapeutic transgene. In the absence of coumarate, rcTA can not be bound to 6xCuO, so the transgenic encoding therapeutic agent will not be transcribed, because 6xCuO is inactive. When coumarate is added, it will form a complex with rcTA, thereby allowing to be bound to 6xCuO and transcribe the transgenic encoding therapeutic agent (Mullick et al., 2006).

[0298] Ecdysone-inducible system

[0299] Another example of an inducible gene expression system is the ecdysone inducible system, in which the N-terminal truncations of the retinoid X receptor (RXR) and the ecdysone receptor (EcR) fused to the activation domain of Vp16 (VpEcR) are inserted into the 5' untranslated region of the gene expressed by the masked cells so that they are co-expressed by the endogenous promoter. An ecdysone response element (EcRE) with a downstream minimal promoter can be inserted just upstream of the start codon of the transgene encoding the therapeutic agent. The co-expressed RXR and VpEcR can heterodimerize with each other. In the absence of ecdysone or the synthetic drug analog muristerone A, the dimerized RXR / VpEcR cannot bind to EcRE and therefore will not transcribe the transgene encoding the therapeutic agent. In the presence of ecdysone or muristerone A, the dimerized RXR / VpEcR can bind to EcRE so that the transgene encoding the therapeutic agent is transcribed (No et al., 1996). Since ecdysone administration has no apparent effects on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene.

[0300] Ligand reversible dimerization system

[0301] In another example, the transgene encoding the therapeutic agent can be modified so that it is functionally divided into parts / domains, such as a 5' part and a 3' part, and the FKBP peptide sequence can be inserted into each domain. An IRES (internal ribosome entry site) sequence can be placed between the two domains, which allows simultaneous transcription of two different domains to produce two separate proteins. In the absence of a dimerizing agent, the two separate domains of the therapeutic agent will be functionally inactive. After the introduction of a dimerizing agent such as rapamycin or AP20187, the FKBP peptide will dimerize, thereby combining the 5' and 3' domains of the therapeutic agent and reconstructing active proteins (Rollins et al., 2000).

[0302] Cell-based delivery of therapeutic agents

[0303] Treatment of age-related macular degeneration or retinal dystrophy

[0304] In one example, the masked cells can be modified to produce a VEGF inhibitor, such as a VEGF trap (e.g., a soluble decoy receptor described in Holash et al., Proc Natl Acad Sci USA 99: 11383-11398, 2002, incorporated herein by reference, such as aflibercept) to treat age-related macular degeneration (AMD) or retinal dystrophy. VEGF traps are biologics that bind to and inhibit VEGF, an angiogenic protein that promotes abnormal blood vessel formation. VEGF traps are used to treat wet AMD, which is characterized by abnormal growth of subretinal blood vessels that can lead to retinal detachment and progressive vision loss. To treat AMD, VEGF traps are typically delivered by regular injection into the eye. Masked cells can be modified to produce a VEGF trap or another VEGF inhibitor by expressing a transgene encoding a VEGF trap or another VEGF inhibitor operably linked to a constitutive or inducible promoter. Can use method known to those skilled in the art before using to eyes to make the cell (for example, stem cell) of the cover expressing VEGF inhibitor (for example, VEGF trap) be differentiated into retinal pigment epithelium (RPE) cell, or can modify the RPE cell of separation to express cover transgene and VEGF inhibitor.Can express VEGF inhibitor (for example VEGF trap) 25,000 to 100,000 cover RPE cells (for example 25,000, 50,000, 75,000 or 100,000 cover RPE cells) be injected into the subretinal space of each eye, to treat wet AMD or retinal dystrophy.Other VEGF inhibitors suitable for compositions and methods described herein include soluble forms (for example soluble VEGFR-1 or NRP-1) of VEGF receptor, platelet factor-4, prolactin, SPARC and VEGF inhibitory antibodies (for example, bevacizumab and ranibizumab).

[0305] Treatment of Parkinson's disease

[0306] In another example, obscured cells, such as dopaminergic neurons or cells (e.g., stem cells) that can be differentiated in vitro to produce dopaminergic neurons using methods known to those skilled in the art, can be administered to a subject with Parkinson's disease (characterized by loss of dopaminergic neurons). 25,000 to 100,000 obscured dopaminergic neurons (e.g., 25,000, 50,000, 75,000, or 100,000 obscured dopaminergic neurons) can be administered (e.g., stereotaxically injected into the substantia nigra) to the brain of a subject with Parkinson's disease.

[0307] Treatment of myocardial infarction

[0308] The masked cells described herein can also be used to treat myocardial infarction (e.g., myocardial infarction, commonly referred to as a heart attack). Myocardial infarction occurs when blood flow is reduced or stopped to a portion of the heart, thereby damaging the myocardium. To treat a subject with myocardial infarction, masked cells (e.g., stem cells) can be differentiated into cardiomyocytes using methods known to those skilled in the art, or isolated cardiomyocytes can be modified to express a masked transgene. Five hundred million to five billion masked cardiomyocytes (e.g., 5 x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , 1x10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 or 5x 10 9 to treat a subject with myocardial infarction (eg, to replace dead or damaged cardiomyocytes).

[0309] Treatment of osteoarthritis and rheumatoid arthritis

[0310] In another example, the masked cells described herein can be used to treat osteoarthritis or rheumatoid arthritis. Osteoarthritis and rheumatoid arthritis (RA) are characterized by inflammation of the joints and are generally treated with anti-inflammatory therapeutics. In order to treat subjects with osteoarthritis or RA, the masked cells can be modified to express anti-inflammatory biologics, such as TNFα inhibitors (e.g., TNFα inhibitors) that have been used clinically for RA treatment. Masked cells can be modified to produce anti-inflammatory biologics, such as TNFα inhibitors, by expressing transgenes encoding anti-inflammatory biologics operably linked to constitutive or inducible promoters. Masked cells (e.g., stem cells) expressing anti-inflammatory biologics (e.g., TNFα inhibitors) can be differentiated into joint fibroblasts using methods known to those skilled in the art before administration to the joint, or separated joint fibroblasts can be modified to express masked transgenes and anti-inflammatory biologics. One million to one hundred million masked joint fibroblasts expressing anti-inflammatory biologics (e.g., 1 x 10 6 , 2x 10 6 , 3x 10 6 , 4x 10 6 , 5x 10 6 , 6x 10 6 , 7x10 6 , 8x10 6 , 9x 10 6 , 1x 10 7, 2x 10 7 , 3x 10 7 , 4x 10 7 , 5x 10 7 , 6x 10 7 , 7x10 7 , 8x 10 7 , 9x 10 7 or 1x 10 8 In some embodiments, the invention relates to the use of a single-cell hybridization system (e.g., a single-cell hybridization system) to treat osteoarthritis or RA. The hybridization system is a process in which a single-cell hybridization system (a single-cell hybridization system) is injected into an arthritic or inflamed joint (depending on the size of the joint) to treat osteoarthritis or RA. Anti-inflammatory biologics that can be expressed by the hybridization cells to treat osteoarthritis or RA include TNFα inhibitors (adalimumab, etanercept, infliximab, golimumab, certolizumab pegol), interleukin-6 (IL6) receptor inhibitors (e.g., tocilizumab), IL1 receptor inhibitors (e.g., anakinra), or other agents used to treat RA (e.g., abatacept, rituximab).

[0311] Treatment of diabetes

[0312] The masked cells can be used to treat diabetes (e.g., type 1 or type 2 diabetes). Type 1 diabetes is caused by the inability of the pancreas to produce enough insulin. Type 2 diabetes begins with insulin resistance, but insulin deficiency may develop as the disease progresses. In order to treat subjects with diabetes, the masked cells can be modified to express insulin, or cells expressing insulin from healthy subjects (e.g., pancreatic β cells from subjects without diabetes) can be modified to express masked transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and SerpinB9 (Spi6) One or more (e.g., one, two, three, four, five, six, seven, or all eight) and administered to subjects with diabetes. The masked cells can be modified to produce insulin by expressing a transgene encoding insulin operably linked to a constitutive or inducible promoter. The masked cells (e.g., stem cells) expressing insulin can be differentiated into insulin-producing cells (e.g., pancreatic β cells) prior to administration using methods known to those skilled in the art or can be administered without differentiation, or isolated pancreatic β cells can be modified to express the masking transgene and optionally express a transgene encoding insulin. Eight to three billion masked pancreatic β cells (e.g., 8 x 10 8 , 9x 10 8 , 1x10 9 , 2x 10 9 or 3x 109 The invention relates to a method for subcutaneously injecting a plurality of masked pancreatic beta cells into a subject to produce masked subcutaneous tissue, which produces insulin for the treatment of diabetes.

[0313] Treatment of hemophilia

[0314] In another example, the masked cells described herein can be used to treat hemophilia. Hemophilia patients do not produce functional factor VIII protein, which is a key blood component required for blood coagulation. These patients may have severe bleeding, and the standard of care includes multiple injections of purified factor VIII protein per week. In order to treat subjects with hemophilia, the masked cells can be modified to express additional transgenics encoding factor VIII. Factor VIII will be constitutively expressed in the masked cells by being operably connected to a constitutive promoter (e.g., CMV or CAG). The masked cells expressing Factor VIII (e.g., stem cells) may be differentiated into cells producing coagulation factors (e.g., sinusoidal cells or endothelial cells) prior to administration using methods known to those skilled in the art or may be administered without differentiation, or isolated sinusoidal cells expressing Factor VIII or endothelial cells from healthy subjects (e.g., subjects without hemophilia) may be modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and SerpinB9 (Spi6) and administered to subjects with hemophilia. If it is necessary to ensure that Factor VIII is expressed at high levels, isolated sinusoidal cells expressing Factor VIII or endothelial cells from healthy subjects that have been modified to express one or more masking transgenes may be further modified to express a transgene encoding Factor VIII. Eight to three billion masked cells (e.g., 8 x 10 8 , 9x 10 8 , 1x 10 9 , 2x 10 9 or 3x 10 9 The invention relates to a method for subcutaneously injecting a plurality of masked cells into a subject to produce masked subcutaneous tissue that produces Factor VIII for the treatment of hemophilia.

[0315] Treatment of metabolic disorders

[0316] The masked cells of the invention may also be used to treat inherited metabolic disorders. In most inherited metabolic disorders, the body does not produce a single enzyme, or it is produced in a defective form. Inherited metabolic disorders include lysosomal storage diseases such as Hurler syndrome (α-L iduronidase deficiency), Niemann-Pick disease (mutations in SMPD1, NPC1, or NPC2), Tay-Sachs disease (mutations in HEXA), Gaucher disease (mutations in the GBA gene), Fabry disease (α-galactosidase deficiency due to mutations in GLA), and Krabbe disease (galactosylceramidase deficiency due to mutations in GALC); galactosemia (galactokinase or galactose-1-phosphate uracil transferase deficiency); maple syrup urine disease (deficiency of the enzyme BCKD); phenylketonuria (deficiency of the enzyme PAH); glycogen storage diseases (GSDs) such as GSD0 (glycogen synthase (GYS2) deficiency), GSD1 / von Gierke's disease (glucose-6-phosphatase (G6PC) deficiency), GSD GSD 2 / Pompe disease (acid alpha-glucosidase (GAA) deficiency), GSD 3 / Cori's disease or Forbes' disease (glycogen debranching enzyme AGL deficiency), GSD 4 / Anderson disease (glycogen branching enzyme (GBE1) deficiency), GSD 5 / McArdledisease (muscle glycogen phosphorylase (PYGM) deficiency), GSD 6 / Hers' disease (liver glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), GSD 7 / Tarui's disease (muscle phosphofructokinase (PKFM) deficiency), GSD 9 (phosphorylase kinase (PHKA2, PHKB, PHKG2 or PHKA1) deficiency), GSD 10 (enolase 3 (ENO3) deficiency), GSD 11 (muscle lactate dehydrogenase (LDHA) deficiency), Fanconi-Bickel syndrome (glucose transporter 2 (GLUT2) deficiency), GSD 12 (aldolase A (ALDOA) ​​deficiency), GSD 13 (β-enolase (ENO3) deficiency), or GSD 15 (glycogenin-1 (GYG1) deficiency);Mitochondrial disorders such as mitochondrial myopathies (Karns-Sell syndrome (KSS, caused by mitochondrial DNA deletions) and chronic progressive external ophthalmoplegia (CPEO, caused by mitochondrial DNA deletions or duplications or mutations in ANT1, POLG, POLG2, or PEO1), diabetes mellitus and deafness (DAD, caused by mutations in mitochondrial DNA at position 3243, which encodes tRNALeu(UUR), Leber hereditary optic neuropathy (LHON, caused by mutations in MT-ND1, MT-ND4, MT-ND4L, and MT-ND6), Leigh syndrome (caused by mutations in SURF1, MT-ATP6, MT-ND2, MT-ND3, MT-ND5, MT-ND6, BCS1L, NDUFA10, SDHA, NDUFS4, NDUFAF2, NDUFA2, NDUFAF6, COX15, ND Mutations in MT-TF, MT-TK, MT-TL1, MT-TH, MT-TS1, MT-TS2, or MT-TF may also cause myopathy, encephalomyopathy, lactic acidosis, and stroke-like symptoms (MELAS, caused by mutations in MT-ND1, MT-ND5, MT-TH, MT-TL1, or MT-TV); Friedreich's ataxia (mutations in FXN); peroxisomal disorders such as Zellweger's syndrome (Zellweger syndrome) (mutations in PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19, or PEX26) and adrenoleukodystrophy (mutations in ABCD1); metal metabolism disorders, such as Wilson disease (mutations in the Wilson disease protein ATP7B) and hemochromatosis (mutations in the human hemochromatosis protein HFE); organic acidemias, such as methylmalonic acidemia (MUT, MMAA, M ; Urea cycle disorders such as ornithine transcarbamylase (OTC) deficiency, arginase (ARG1) deficiency, argininosuccinate lyase deficiency (ASL) deficiency, argininosuccinate synthase 1 (ASS1) deficiency, citrin deficiency, carbamoyl phosphate synthase 1 (CPSI) deficiency, N-acetylglutamate synthase (NAGS) deficiency, and ornithine translocase (ORNT1) deficiency.

[0317] To treat a subject with a metabolic disorder, the masked cells can be modified to express a wild-type form of a gene mutated in the subject or a transgene encoding an enzyme that is missing or deficient in the subject (see Table 2), or cells from a healthy subject (e.g., a subject not suffering from a metabolic disorder) expressing a wild-type form of a gene mutated in the subject or an enzyme that is deficient in the subject can be modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the masked transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject with a metabolic disorder. The wild-type form of a gene mutated in the subject or a transgene encoding an enzyme that is missing or deficient in the subject can be constitutively expressed in the masked cells by being operably linked to a constitutive promoter (e.g., CMV or CAG) or can be inducibly expressed using one of the inducible expression systems described herein. Masked cells (e.g., stem cells) modified to express wild-type forms of genes mutated in a subject or enzymes deleted or deficient in a subject can be differentiated into cells that normally express the gene or enzyme using methods known to those skilled in the art before administration or can be administered without differentiation, or isolated cells expressing wild-type forms of the gene or enzymes mutated or deficient in a subject from healthy subjects can be modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the masking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject with a metabolic disorder. If the subject has not been diagnosed with a specific mutation prior to treatment, the subject can be evaluated using standard methods to identify mutated genes associated with the metabolic disorder to ensure that the masked cells express the corresponding wild-type gene. Eighty to three billion masked cells (e.g., 8 x 10 8 , 9x 10 8 , 1x 10 9 , 2x 10 9 or 3x 10 9 masked cells) to generate masked subcutaneous tissue that produces the corresponding wild-type protein.

[0318] Methods for controlling the division of masked cells

[0319] In one aspect, a method of controlling proliferation of cells at a transplantation site in an allogeneic host is provided (eg, reducing the tumorigenic potential of cells at the transplantation site or reducing proliferation of cells at the transplantation site that have become tumorigenic).

[0320] The method comprises: providing a cell or a population of such cells that is genetically modified to contain at least one mechanism for providing local immunosuppression at the site of transplantation when transplanted into an allogeneic host; genetically modifying a cell division locus / multiple cell division loci (CDL) in the cell, wherein the CDL is one or more loci whose transcription products are expressed by dividing cells (e.g., all dividing cells contain one or more of the immunosuppressive transgenes), wherein the genetic modification of the CDL comprises one or more of the following: a) an Eliminate Link (ALINK) system, wherein the ALINK system comprises a DNA sequence encoding a negative selectable marker transcriptionally linked to a DNA sequence encoding the CDL; and b) a CDL-regulated inducible exogenous activator (EARC) system, wherein the EARC system comprises an inducible activator-based gene expression system operably linked to the CDL; The invention relates to a method for producing a cell modified by the ALINK system and a method for producing a cell modified by the ALINK system. The method comprises: maintaining the genetically modified cells comprising the ALINK system in the absence of an inducer of the negative selective marker to allow the proliferation of the genetically modified cells comprising the ALINK system, and / or eliminating and / or inhibiting the proliferation of the cells comprising the ALINK system by exposing the genetically modified cells comprising the ALINK system to an inducer of the gene expression system based on the inducible activating factor; and / or allowing the proliferation of the genetically modified cells comprising the EARC system by exposing the genetically modified cells comprising the EARC system to an inducer of the gene expression system based on the inducible activating factor, or preventing or inhibiting the proliferation of the cells comprising the EARC system by maintaining the genetically modified cells comprising the EARC system in the absence of an inducer of the gene expression system based on the inducible activating factor; and transplanting the cells or cell populations at a transplantation site in an allogeneic host. Cells modified in one or more CDLs (e.g., 2, 3, 4 or more CDLs) using one or more ALINK and / or EARC systems to control cell division can be referred to as "fail-safe cells." The number of cells (e.g., clonal capacity) that can grow before a single fail-safe cell loses the activity of all systems (e.g., ALINKs or EARCs) that control cell division through genetic mutation (e.g., based on mathematical modeling, the number of cell divisions required for a cell to "escape" from control and exhibit uncontrollable cell proliferation) determines the fail-safe capacity. The fail-safe capacity will depend on the number of ALINKs and the number of CDLs targeted by the ALINKs. The fail-safe properties are further described in Table 3.

[0321] Table 3. Mathematical modeling was used to calculate the fail-safe cell capacity and its relevance to humans. The model does not take into account events where CDL expression is lost together with the loss of negative selective marker activity, thereby impairing cell proliferation. Therefore, the values ​​are underestimated and 10% of negative selective marker activity is assumed. 6 The estimated number of cells in the human body is 3.72x10 13 Adapted from (Bianconi et al., 2013).

[0322]

[0323] In various embodiments, the CDL is a locus identified as an "essential gene" as described in Wang et al., 2015 (which is incorporated herein by reference in its entirety). Essential genes in Wang et al., 2015 are identified by calculating a score (i.e., a CRISPR score) for each gene that reflects the fitness cost imposed by the inactivation of the gene. In one embodiment, the CDL has a CRISPR score (CS) of less than about -1.0 (Table 5, column 5).

[0324] In various embodiments, the CDL is a locus / locus encoding a gene product associated with cell division and / or replication (Table 5, column 6). For example, in various embodiments, the CDL is a locus / locus encoding a gene product associated with one or more of the following: i) cell cycle; ii) DNA replication; iii) RNA transcription and / or protein translation; and iv) metabolism (Table 5, column 7).

[0325] In one embodiment, CDL is one or more cyclin-dependent kinases involved in regulating the progression of the cell cycle (e.g., controlling G1 / S G2 / M and metaphase to anaphase transitions), such as CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9 and / or CDK11 (Morgan, 2007). In one embodiment, CDL is one or more cyclins involved in controlling the progression of the cell cycle by activating one or more CDKs, such as, for example, cyclin B, cyclin E, cyclin A, cyclin C, cyclin D, cyclin H, cyclin C, cyclin T, cyclin L and / or cyclin F (FUNG and POON, 2005). In one embodiment, CDL is one or more loci involved in the anaphase promoting complex, which controls the progression of the metaphase to anaphase transition in the M phase of the cell cycle (Peters, 2002). In one embodiment, the CDL is one or more loci involved in centromere components that control the progression of the metaphase to anaphase transition in the M phase of the cell cycle (Fukagawa, 2007). In one embodiment, the CDL is one or more loci involved in microtubule components that control microtubule dynamics required for the cell cycle (Cassimeris, 1999).

[0326] In various embodiments, the CDL is a locus / locus involved in housekeeping. As used herein, the term "housekeeping gene" or "housekeeping locus" refers to one or more genes required to maintain basic cellular functions. Housekeeping genes are expressed in all cells of an organism under normal and patho-physiological conditions.

[0327] In various embodiments, a CDL is a locus / locus encoding a gene product associated with cell division and / or proliferation and having a CRISPR score of less than about -1.0. For example, in one embodiment, a CDL is a locus / locus encoding a gene product associated with one or more of: i) cell cycle; ii) DNA replication; iii) RNA transcription and / or protein translation; and iv) metabolism and having a CRISPR score of less than about -1.0. In one embodiment, a CDL may also be a housekeeping gene.

[0328] In some embodiments, the CDL is Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CEPNA, Birc5 / BIRC5, or Eef2 / EEF2. In some embodiments, the CDL is Cdk1 / CDK1. In some embodiments, the CDL is Top2A / TOP2A. In some embodiments, the CDL is Eef2 / EEF2. In some embodiments, the CDL is Cdk1 / CDK1 and Top2A / TOP2A or Cdk1 / CDK1 and Eef2 / EEF2.

[0329] By linking the expression of CDL with the expression of a DNA sequence encoding a negative selective marker, cells can be modified to "fail-safe" cells, thereby allowing drug-induced elimination of mitotically active cells expressing both CDL and negative selective markers. For example, when cell proliferation is uncontrolled and / or accelerated relative to the normal division rate of cells (e.g., uncontrolled cell division exhibited by cancer cells), or when the therapeutic needs of cells have passed, it may be desirable to eliminate proliferating cells. The elimination of proliferating cells can be achieved via genetic modification of cells, referred to herein as "elimination connection" (ALINK), which links the expression of a DNA sequence encoding a negative selective marker with the expression of CDL, thereby allowing the elimination or full inhibition of proliferating cells modified by ALINK, thereby expressing the CDL locus (full inhibition is to inhibit the cell proliferation rate to a rate that is too low to promote tumor formation). In the presence of a prodrug or other inducer of a negative selective system, cells expressing a negative selective marker will stop proliferating or die, depending on the mechanism of action of the selective marker. Cells can be modified to contain homozygous, heterozygous, hemizygous, or compound heterozygous ALINK. In one embodiment, to improve the fidelity of elimination, negative selectable markers may be introduced into all functional alleles of CDL. In a preferred embodiment, negative selectable markers may be introduced into all functional alleles of CDL. If desired, a fail-safe system may be used to eliminate all obscured cells.

[0330] ALINK can be inserted at any position of CDL, which allows co-expression of CDL and a negative selectable marker.

[0331] In some embodiments, the ALINK system comprises a herpes simplex virus-thymidine kinase / ganciclovir system, a cytosine deaminase / 5-fluorocytosine system, a carboxylesterase / irinotecan system, or an iCasp9 / AP1903 system.

[0332] DNA encoding a negative selectable marker (e.g., HSV-TK) can be inserted into a CDL (e.g., CDK1) in a host cell so that expression of the negative selectable marker causes the host cell to express the negative selectable marker and, necessarily, causes the CDL to be killed in the presence of an inducer (e.g., a prodrug) of the negative selectable marker (e.g., ganciclovir (GCV)). In this example, host cells modified with ALINK will produce thymidine kinase (TK), and the TK protein converts GCV to GCV monophosphate, which is then converted to GCV triphosphate by cellular kinases. GCV triphosphate is incorporated into replicating DNA during the S phase, which leads to termination of DNA elongation and apoptosis (Halloran and Fenton, 1998).

[0333] A modified HSV-TK gene (Preuβ et al., 2010) is disclosed herein as an example of a DNA encoding a negative selectable marker that can be used in ALINK genetic modification to selectively eliminate cells comprising an undesirable cell division rate.

[0334] It is contemplated herein that alternative and / or additional negative selectivity systems may be used in the tools and / or methods provided herein. Various negative selectivity marker systems are known in the art (eg, dCK.DM (Neschadim et al., 2012)).

[0335] For example, in the field of "gene-directed enzyme / prodrug therapy" (GEPT), various negatively selective systems with clinical relevance have been actively developed with the goal of improving the therapeutic efficacy of conventional cancer therapies with no or minimal side effects (Hedley et al., 2007; Nawa et al., 2008). Typically, GEPT involves the use of viral vectors to deliver genes into or near cancer cells in areas of mammalian cells where the cancer cell is not found and produces enzymes that convert relatively nontoxic prodrugs into toxic agents.

[0336] HSV-TK / GCV, cytosine deaminase / 5-fluorocytosine (CD / 5-FC), and carboxylesterase / irinotecan (CE / CPT-11) are examples of negative selective marker systems evaluated in preclinical and clinical trials for GEPT (Danks et al., 2007; Shah, 2012).

[0337] To overcome the potential immunogenicity of the herpes simplex virus type 1 thymidine kinase / ganciclovir (TK / GCV) system, a “humanized” suicide system has been developed by engineering human deoxycytidine kinase to become thymidine active and used as a negatively selective (suicide) system antibody with a non-toxic prodrug: bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), or L-deoxyuridine (LdU) (Neschadim et al., 2012).

[0338] The CD / 5-FC negative selective marker system is a widely used "suicide gene" system. Cytosine deaminase (CD) is a non-mammalian enzyme that can be obtained from bacteria or yeast (e.g., from Escherichia coli or Saccharomyces cerevisiae, respectively) (Ramnaraine et al., 2003). CD catalyzes the conversion of cytosine to uracil and is an important member of the pyrimidine salvage pathway in prokaryotes and fungi, but it does not exist in mammalian cells. 5-Fluorocytosine (5-FC) is an antifungal prodrug that causes lower levels of cytotoxicity in humans (Denny, 2003). CD catalyzes the conversion of 5-FC to the genotoxic agent 5-FU, which has high levels of toxicity in humans (Ireton et al., 2002).

[0339] The CE / CPT-11 system is based on carboxylesterases, which are serine esterases found in different tissues of mammalian species (Humerickhouse et al., 2000). The anticancer agent CPT-11 is a prodrug activated by CE to produce an active species called 7-ethyl-10-hydroxycamptothecin (SN-38), which is a potent mammalian topoisomerase I inhibitor (Wierdl et al., 2001). SN-38 induces accumulation of double-stranded DNA breaks in dividing cells (Kojima et al., 1998).

[0340] Another example of a negative selective marker system is the iCasp9 / AP1903 suicide system, which is based on a modified human caspase 9 fused to human FK506 binding protein (FKBP) to allow chemical dimerization using the small molecule AP1903, which has been tested for safety in humans. Administration of the dimerized drug induces apoptosis of cells expressing the engineered caspase 9 component. This system has several advantages, such as, for example, including low potential immunogenicity, because it is composed of human gene products, so the dimer drug only affects cells expressing the engineered caspase 9 component (Straathof et al., 2005). The iCasp / AP1903 suicide system is being tested in a clinical setting (Di Stasi et al., 2011).

[0341] It is contemplated herein that the negative selective marker system of the ALINK system may be replaced by a proliferation antagonist system. As used herein, the term "proliferation antagonist" refers to a natural or engineered compound whose presence inhibits (completely or partially) cell division. For example, Omomyc ER It is a fusion protein of the dominant negative MYC Omomyc and the mutant mouse estrogen receptor (ER) domain. When induced by tamoxifen (TAM), the fusion protein Omomyc ER Localized to the nucleus, where the dominant negative Omomyc dimerizes with C-Myc, L-Myc, and N-Myc, thereby binding them in a complex that cannot bind to the Myc DNA binding consensus sequence (Soucek et al., 2002). Due to the lack of Myc activity, cells cannot divide (Oricchio et al., 2014). Another example of a proliferation antagonist is A-Fos, which is a dominant negative activating protein-1 (AP1) (a heterodimer of the oncogene Fos and Jun) that inhibits DNA binding in equimolar competition (Olive et al., 1997). A-Fos can also be fused to the ER domain, making its nuclear localization induced by TAM. Omomyc ER / Tamoxifen or A-Fos ER / Tamoxifen can be a replacement for TK / GCV to become ALINK.

[0342] By operably connecting CDL to EARC (e.g., a gene expression system based on an inducible activator), cells can also be modified to be "fail-safe". Under these conditions, CDL will only be expressed in the presence of an inducing agent of a gene expression system based on an inducible activator (and the cells can only divide). Under these conditions, depending on the mechanism of action of the gene expression system based on an inducible activator and the CDL function, in the absence of an inducing agent, the EARC-modified cells stop dividing, significantly slow down, or die. Cells can be modified to include homozygous or compound heterozygous EARCs, or changes can be made so that only EARC-modified alleles can produce functional CDLs. In one embodiment, for example, EARC modifications can be introduced into all alleles of CDL to provide a mechanism for cell division control.

[0343] The EARC can be inserted into the CDL at any position that allows co-expression of the CDL and the activator component of the inducible system in the presence of an inducing agent.

[0344] In one embodiment, an "activator" based gene expression system is preferred over a "repressor" based gene expression system. For example, if a repressor is used to suppress CDL, a loss of repressor function mutation can release CDL expression, allowing cells to proliferate. In activation-based suppression of cell division, loss of activator function (mutation) will shut down CDL expression, thereby preventing cell proliferation.

[0345] In some embodiments, the EARC system is a dox-bridge system, a coumarate switch inducible system, an ecdysone inducible system, a radio wave inducible system, or a ligand reversible dimerization system.

[0346] The dox-bridge can be inserted into a CDL (e.g., CDK1) in a host cell so that in the presence of an inducer (e.g., doxycycline or "DOX"), the dox-bridge allows CDL expression, thereby allowing cell division and proliferation. Host cells modified with dox-bridge EARCs may contain a reverse tetracycline transactivator (rtTA) gene (Urlinger et al., 2000) under the transcriptional control of a promoter, which is active in dividing cells (e.g., in CDL). This targeted insertion makes the CDL promoter no longer available for CDL transcription. In order to restore CDL transcription, a tetracycline response element promoter (e.g., TRE (Agha-Mohammadi et al., 2004)) is inserted before the CDL transcript, which will only express the CDL gene in the presence of rtTA and doxycycline. When the only source of CDL expression is the dox-bridged allele, there is no CDL gene expression in the absence of doxycycline. The lack of CDL expression causes EARC-modified cells to be impaired in their proliferation, either by dying, arresting cell division, or causing the cell mitosis rate to become so slow that the EARC-modified cells cannot contribute to tumor formation.

[0347] As used herein, the term "dox-bridge" refers to a mechanism that separates the activity of the promoter from the target transcription region and realizes the transcription of the target region under the control of TRE by expressing rtTA (Gossen et al., 1995) from an endogenous or exogenous promoter. As used herein, "rtTA" refers to the reverse tetracycline transactivator element (Gossen et al., 1995) of the tetracycline inducible system, and "TRE" refers to a promoter consisting of the TetO operon sequence upstream of the minimal promoter. After the binding of rtTA to the TRE promoter in the presence of doxycycline, the transcription of the locus downstream of the TRE promoter increases. The rtTA sequence can be inserted into the same transcription unit as CDL, or into different locations of the genome, as long as the permissible or nonpermissible state of the transcriptional expression of the target region is controlled by doxycycline. Dox-bridge is an example of EARC.

[0348] This paper also considers introducing the EARC system into the 5' regulatory region of CDL.

[0349] It is contemplated herein that alternative and / or additional inducible activator-based gene expression systems may be used in the tools and / or methods provided herein to generate EARC modifications. Various inducible activator-based gene expression systems are known in the art.

[0350] For example, when cell division and / or proliferation is required, a destabilizing protein domain fused to the effector protein product encoding CDL (Banaszynski et al., 2006) can be used in combination with a small molecule synthetic ligand to stabilize the CDL fusion protein. In the absence of a stabilizer, the unstable CDL protein will be degraded by the cell, which will then stop proliferation. When a stabilizer compound is added, it will bind to the unstable CDL protein, which will not be degraded, thereby allowing cell proliferation.

[0351] For example, transcription activator-like effector (TALE) technology (Maeder et al., 2013) can be combined with dimer-regulated expression induction (Pollock and Clackson, 2002). TALE technology can be used to generate DNA binding domains designed to be specific to a sequence and placed together with a minimal promoter that replaces the CDL promoter. The TALE DNA binding domain is also extended with a drug dimerization domain. The latter can bind to another engineered protein with a corresponding dimerization domain and a transcriptional activation domain.

[0352] For example, reverse-coumarate-transactivator (rcTA) can be inserted in the 5' untranslated region of CDL so that it will be expressed by the endogenous CDL promoter. Six times of repetition (6xCuO) of the coumarate operon can be inserted just before the translation initiation (ATG) of CDL. In the absence of coumarate in the system, rcTA cannot be bound to 6xCuO, so CDL will not be transcribed because 6xCuO is inactive. When coumarate is added, it will form a complex with rcTA, thereby achieving binding with 6xCuO and achieving CDL transcription (Mullick et al., 2006).

[0353] For example, N-terminal truncations of the retinoid X receptor (RXR) and ecdysone receptor (EcR) fused to the activation domain of Vp16 (VpEcR) can be inserted into the 5' untranslated region of CDL so that they are co-expressed by the endogenous CDL promoter. An ecdysone response element (EcRE) with a downstream minimal promoter can also be inserted into CDL just upstream of the start codon. The co-expressed RXR and VpEcR can heterodimerize with each other. In the absence of ecdysone or the synthetic drug analog melesterone A, the dimerized RXR / VpEcR cannot bind to the EcRE, so the CDL is not transcribed. In the presence of ecdysone or melesterone A, the dimerized RXR / VpEcR can bind to the EcRE so that the CDL is transcribed (No et al., 1996).

[0354] For example, transient receptor potential vanilloid-1 (TRPV1) can be inserted into the 5' untranslated region of CDL along with ferritin and co-expressed by the endogenous CDL promoter. A promoter inducible by NFAT (NFATre) can also be inserted into CDL just upstream of the start codon. Under normal circumstances, the NFAT promoter is inactive. However, after exposure to low-frequency radio waves, TRPV1 and ferritin produce Ca2+ that enters the cell. ++ The NFATc converts cytoplasmic NFAT into nuclear NFAT, which eventually activates NFATre and transcribes CDL (Stanley et al., 2015).

[0355] For example, CDL can be functionally divided into multiple parts / domains: 5'-CDL and 3'CDL, and the FKBP peptide sequence can be inserted into each domain. An IRES (internal ribosome entry site) sequence can be placed between the two domains, which will be transcribed simultaneously by the CDL promoter, but two separate proteins will be produced. In the absence of an inducer, the two separate CDL domains will be functionally inactive. Upon introduction of a dimerizing agent such as rapamycin or AP20187, the FKBP peptide will dimerize, thereby binding the 5' and 3' CDL parts together and reconstructing the active protein (Rollins et al., 2000).

[0356] In one embodiment of the method, the genetically modified cell comprises: a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane-bound or locally acting and whose function is to reduce the function of the host immune system (e.g., leukocytes attacking the transplant and NK cell activation) or serve as a defense mechanism against attacking leukocytes.

[0357] When transplanting cells or populations of such cells in an allogeneic host, methods for genetically modifying the cells to comprise at least one mechanism for providing local immunosuppression at the transplantation site are described, for example, in WO 2016 / 141480, the entire teachings of which are incorporated herein by reference.

[0358] The group of transgenes includes one or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). In one embodiment, the group of transgenes includes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6).

[0359] Optionally, the method further comprises expressing one or more of the following transgenes in the cell: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39. In one embodiment, the TGF-β or biologic acts locally in the transplant environment.

[0360] Techniques for introducing various genetic modifications, such as transgenes, into animal cells are described herein and generally known in the art.

[0361] In one embodiment of the method, the cell is a stem cell, a cell suitable for genome editing, or a cell that can serve as a source of a therapeutic cell type (for example, a cell that can be directed to differentiate into a lineage-restricted or terminally differentiated cell that can be used for cell therapy, or a cell of a desired target tissue). In one embodiment, the cell is an embryonic stem cell, an induced pluripotent stem cell, an adult stem cell, a tissue-specific stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem cell or a progenitor cell, a germline stem cell, a lung stem cell or a progenitor cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, a pluripotent stem cell, an amniotic membrane stem cell, an umbilical cord blood stem cell, or a neural stem cell or a progenitor cell. In some embodiments, the cell is derived from a target tissue, such as skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach. In some embodiments, the cell is a fibroblast, an epithelial cell or an endothelial cell. The cell can be a vertebrate cell, such as a mammalian cell, such as a human or mouse cell.

[0362] Techniques for transplanting genetically modified cells into an allogeneic host at a transplantation site are described herein and generally known in the art.

[0363] In various embodiments of any of the methods of the present disclosure, the host suffers from a degenerative disease or condition that can be treated with cell therapy. Examples of such diseases or conditions include, but are not limited to, blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, nervous system disease (e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS), enzyme or hormone deficiency, metabolic disorders (e.g., lysosomal storage disease, galactosemia, maple syrup urine disease, phenylketonuria, glycogen storage disease, mitochondrial disease, Friedreich's ataxia, peroxisomal disease, metal metabolism disorders or organic acidemias, autoimmune diseases (e.g., psoriasis, systemic lupus erythematosus, Graves' disease, inflammatory bowel disease, Addison's disease, Sjögren's syndrome), Hashimoto's thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune immune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infectious disease, hemophilia, degenerative disease (e.g., Charcot-Marie-Doucet disease, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, cystic fibrosis, cytochrome C oxidase deficiency, Ehlers-Danlos syndrome, essential tremor, fibrodysplasia ossificans progressiva, infantile neuroaxonal dystrophy, keratoconus, keratoglobose, muscular dystrophy, neuronal ceroid lipofuscinosis, pre-existing disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa), or age-related disease (e.g., atherosclerosis, cardiovascular disease (e.g., angina, myocardial infarction), cataract, osteoporosis, or hypertension).

[0364] Pharmaceutical composition

[0365] The masked cells described herein can be incorporated into a vehicle for administration to a patient, such as a human patient receiving a transplant or suffering from a disease or disorder described herein. Pharmaceutical compositions containing masked cells can be prepared using methods known in the art. For example, such compositions can be prepared using, for example, a physiologically acceptable carrier, excipient, or stabilizer (Remington: The Science and Practice of Pharmacology 22nd Edition, Allen, L. Ed. (2013); which is incorporated herein by reference) and in a desired form, such as in the form of an aqueous solution.

[0366] The cells covered as described herein can be applied in any physiologically compatible carrier such as a buffered saline solution. Pharmaceutically acceptable carriers and diluents include saline, buffered aqueous solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Other examples include liquid culture media, such as Dulbecco's modified Eagle's medium (DMEM), sterile saline, sterile phosphate buffered saline, Leibovitz's medium (L15, Invitrogen, Carlsbad, Calif.), dextrose in sterile water, and any other physiologically acceptable liquid. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. The carrier can be a solvent or dispersion medium containing the following: for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. For example, by using a coating such as lecithin, in the case of a dispersion, by maintaining the desired particle size and by using a surfactant to maintain appropriate fluidity. In many cases, it is preferred to include an isotonic agent, such as sugar or sodium chloride. The solution is preferably sterile and fluid to the extent that easy syringability exists. Preferably, the solution is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi by using, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. The solutions of the present invention can be prepared by using a pharmaceutically acceptable carrier or diluent and, as required, other ingredients listed above, followed by filter sterilization and then incorporating the masked cells as described herein.

[0367] For example, if necessary, a solution containing a pharmaceutical composition as described herein can be appropriately buffered, and the liquid diluent can first be made isotonic with sufficient saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, usable sterile aqueous media will be known to those skilled in the art in light of the present disclosure. The individual responsible for administration will determine the appropriate dose for individual subjects in any case. In addition, for human administration, the preparation can meet the requirements of the FDA office for sterility, pyrogenicity, general safety and purity standards for biological product standards.

[0368] The pharmaceutical composition comprising the masked cells in a semi-solid or solid carrier is typically formulated for surgical implantation at a transplant site or an affected site of a disease or disorder in a subject. It should be understood that liquid compositions may also be administered by surgical procedures. In particular embodiments, the semi-solid or solid pharmaceutical composition may comprise a semi-permeable gel, matrix, cell scaffold, etc., which may be non-biodegradable or biodegradable. For example, in certain embodiments, it may be desirable or appropriate to isolate the masked cells from their surroundings, but still allow the cells to secrete biomolecules and deliver the biomolecules (e.g., therapeutic agents listed in Table 2) to surrounding cells.

[0369] In other embodiments, different types of degradable gels and networks are used in the pharmaceutical compositions of the present invention. For example, degradable materials include biocompatible polymers such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.

[0370] In another embodiment, one or more hydrogels are used for pharmaceutical compositions. One or more hydrogels may include collagen, atelocollagen, fibrin constructs, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate and poly (ethylene oxide). In addition, hydrogels may be formed by poly (2-hydroxyethyl methacrylate), poly (acrylic acid), self-assembling peptides (e.g. RAD16), poly (methacrylic acid), poly (N-vinyl-2-pyrrolidone), poly (vinyl alcohol) and copolymers thereof with each other and with hydrophobic monomers such as methyl methacrylate, vinyl acetate, etc. It is also preferred that the hydrophilic polyurethane containing large poly (ethylene oxide) blocks. Other preferred materials include hydrogels comprising an interpenetrating network structure of a polymer formed by addition or by polycondensation, and the components of the polymer may include hydrophilic and hydrophobic monomers, such as those monomers just cited. Degradable networks formed in situ are also suitable for the present invention (see, e.g., Anseth, KS et al. J. Controlled Release, 2002; 78: 199-209; Wang, D. et al., Biomaterials, 2003; 24: 3969-3980; U.S. Patent Publication 2002 / 0022676). These in situ formed materials are formulated into fluids suitable for injection; the formation of hydrogels can then be induced by a variety of means such as changes in temperature, pH, and exposure to light in situ or in vivo. In one embodiment, the construct comprises a gel containing fibrin glue. In another embodiment, the construct comprises a gel containing atelocollagen.

[0371] The polymer used to form the matrix can be in the form of a hydrogel. Generally, a hydrogel is a cross-linked polymer material that can absorb more than 20% of its weight in water while maintaining a unique three-dimensional structure. This definition includes dry cross-linked polymers and water-swollen materials that will swell in an aqueous environment. Whether the polymer is biologically derived, semi-synthetic or fully synthetic, many hydrophilic polymers can be cross-linked to produce a hydrogel. The hydrogel can be produced by a synthetic polymer material. Such synthetic polymers can be customized to have a certain range of properties and predictable batch consistency, and represent a reliable source of materials that usually do not need to worry about immunogenicity. The matrix can include a hydrogel formed by self-assembling peptides, such as those discussed in U.S. Patent Nos. 5,670,483 and 5,955,343, U.S. Patent Application No. 2002 / 0160471 and PCT Application No. WO 02 / 062969.

[0372] The properties that make hydrogels valuable in drug delivery applications include equilibrium swelling, adsorption kinetics, solute permeability and in vivo performance characteristics. The permeability of the compound depends in part on the swelling or water content and the biodegradation rate. Since the mechanical strength of the gel may decrease in proportion to the swelling, it is also within the scope of the present invention that the hydrogel can be attached to a substrate so that the composite system improves the mechanical strength. In some embodiments, the hydrogel can be impregnated in a porous substrate to obtain the mechanical strength of the substrate and the useful delivery properties of the hydrogel.

[0373] In other embodiments, the pharmaceutical composition comprises a biocompatible matrix made of natural, modified natural, or synthetic biodegradable polymers, including homopolymers, copolymers, and block polymers, and combinations thereof.

[0374] Examples of suitable biodegradable polymers or polymer classes include any biodegradable polymer discussed in the present disclosure, including but not limited to fibrin, collagen types I, II, III, IV and V, elastin, gelatin, vitronectin, fibronectin, laminin, thrombin, poly(amino acids), oxidized cellulose, tropoelastin, filamentous fibers, ribonucleic acids, deoxyribonucleic acids, proteins, polynucleotides, gum arabic, recombinant basement membrane matrix, starch, dextran, alginate, hyaluronic acid, chitin, chitosan, agarose, polysaccharides, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polyethylene glycol, decellularized tissues, self-assembling peptides, polypeptides, glycosaminoglycans, their derivatives and mixtures thereof. Suitable polymers also include poly(lactide) (PLA), polyhydroxybutyrate, polyurethane, polyphosphazene, poly(ethylene glycol)-poly(lactide-co-glycolide) copolymers, degradable polycyanoacrylates and degradable polyurethanes that can be formed from L(+) and D(-) polymers. For both glycolic acid and lactic acid, intermediate cyclic dimers can be prepared and purified prior to polymerization. These intermediate dimers are called glycolide and lactide, respectively.

[0375] Other useful biodegradable polymers or polymer classes include, but are not limited to, aliphatic polyesters, poly(alkylene oxalates), tyrosine-derived polycarbonates, polyiminocarbonates, polyorthoesters, polyoxaesters, polyamide polyesters, polyoxaesters containing amine groups, poly(propylene fumarate), polyfumarates, polydioxanones, polycarbonates, polyoxalates, poly(α-hydroxy acids), poly(esters), polyurethanes, poly(ester urethanes), poly(ether urethanes), polyanhydrides, polyacetates, polycaprolactones, poly(orthoesters), polyamino acids, polyamides, and blends and copolymers thereof. Additional useful biodegradable polymers include, but are not limited to, stereopolymers of L- and D-lactic acid, copolymers of bis(p-carboxyphenoxy)propane and sebacic acid, copolymers of sebacic acid, copolymers of caprolactone, poly(lactic acid) / poly(glycolic acid) / polyethylene glycol copolymers, copolymers of polyurethane and polylactic acid, copolymers of α-amino acids, copolymers of α-amino acids and caproic acid, copolymers of α-glutamic acid benzyl ester and polyethylene glycol, copolymers of succinate and poly(glycol), polyphosphazenes, poly(hydroxyalkanoates), and mixtures thereof. Binary and ternary systems are also contemplated.

[0376] In general, the material used to form the matrix is ​​ideally configured so that it: (1) has mechanical properties suitable for the intended application; (2) remains sufficiently intact until tissue ingrowth and healing occurs; (3) does not induce an inflammatory or toxic response; (4) is metabolized in vivo after its purpose has been achieved; (5) is easily processed into the desired end product to be formed; (6) exhibits an acceptable shelf life; and (7) is easily sterilized.

[0377] In another embodiment, the cell colony of covering can be applied by using a support. The composition, shape and porosity of the support can be any of the above. Usually, these three-dimensional biomaterials contain living cells attached to the support, dispersed in the support or incorporated into the extracellular matrix embedded in the support. Once implanted in the target area of ​​the body, these implants become integrated with the host tissue, and the transplanted cells gradually become established.

[0378] Non-limiting examples of scaffolds that can be used include fabric structures such as woven fabrics, knitted fabrics, braided fabrics, mesh fabrics, nonwovens, and warp knitted fabrics; porous foams, semi-porous foams, perforated films or sheets, microparticles, beads and balls, and composite structures that are combinations of the above structures. Nonwoven pads can be formed, for example, using fibers composed of synthetic absorbable copolymers of glycolic acid and lactic acid (PGA / PLA), which are sold under the trade name VICRYL sutures (Ethicon, Inc., Somerville, NJ). Foams composed of, for example, poly(ε-caprolactone) / poly(glycolic acid) (PCL / PGA) copolymers, formed by methods such as freeze drying or lyophilization, as discussed in U.S. Pat. No. 6,355,699, can also be used.

[0379] In another embodiment, the framework is a felt, which can be composed of multifilaments made from bioabsorbable materials. Standard textile processing techniques including crimping, cutting, carding and needle punching can be used to make the yarn into a felt. In another embodiment, cells are seeded onto a foam scaffold that can be used as a composite structure.

[0380] The framework can be molded into a useful shape so as to fill tissue voids. Thus, the framework can be shaped to provide not only a channel for nerve growth, but also a scaffold for supporting and surrounding tissue (such as vascular tissue, muscle tissue, etc.). In addition, it will be understood that the cell population can be cultured on a preformed, non-degradable surgical or implantable device.

[0381] Pharmaceutical compositions may include preparations made from masked cells formulated with a pharmaceutically acceptable carrier or medium. Suitable pharmaceutically acceptable carriers include any carrier discussed in the present disclosure, including but not limited to water, saline solutions (such as Ringer's solution), alcohols, oils, gelatin, polyvinylpyrrolidine, carbohydrates (such as lactose, amylose or starch), fatty acid esters and hydroxymethylcellulose. Such preparations may be sterilized and, if necessary, may be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers and colorants. Pharmaceutical carriers suitable for use in the present invention are known in the art and are described in, for example, Pharmaceutical Sciences (17th edition, Mack Pub. Co., Easton, Pa.) and WO 96 / 05309.

[0382] Treatment

[0383] The masked cells and compositions described herein can be administered to a subject in need (e.g., a subject who is receiving or has received a transplant, or a subject suffering from a disease or disorder described herein) by a variety of routes, such as local administration to or near the site of transplantation, local administration to the site affected by the disease or disorder (e.g., injection into a joint for the treatment of RA, injection into the subretinal space for the treatment of wet AMD, direct administration to the central nervous system (CNS) (e.g., intracerebral, intraventricular, intrathecal, intracisternal or stereotactic administration) for the treatment of neurological diseases such as Parkinson's disease, direct injection into the myocardium for the treatment of myocardial infarction), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, infusion, lavage, and oral administration. The most appropriate route of administration in any given case will depend on the specific cells or compositions being administered, the patient, the method of drug formulation, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate. The composition can be administered once or more than once (e.g., once a year, twice a year, three times a year, twice a month, or once a month). For local administration, the masked cells can be administered by any means that places the cell population in the desired location, including catheters, syringes, shunts, stents, microcatheters, pumps, implanted with a device, or implanted with a stent.

[0384] As described herein, before administration, the cell colony can be hatched under the conditions of one or more factors or under the conditions of stimulating stem cell differentiation into desired cell type (for example, neuron, cardiomyocyte, RPE cell, cell producing insulin, cell producing coagulation factor, articular fibroblast or other cell types described herein). Such factors are known in the art, and those skilled in the art will appreciate that suitable differentiation conditions can be determined by routine experiments. Such factors include growth or trophic factors, chemokines, cytokines, cell products, demethylating agents and other stimulants known for example along angiogenesis, hemangiogenic, vasculogenic, skeletal muscle, vascular smooth muscle, pericytes, neurons or vascular endothelial pathways or pedigrees to stimulate the differentiation of stem cells. Alternatively, the composition applied to the patient comprises a cell colony covered with one or more factors stimulating cell differentiation into desired cell type, wherein cell differentiation occurs in vivo at tissue site. In some embodiments, methods known to those skilled in the art can be used to make the cell differentiation of covering into organ or tissue in vitro, and applied to the subject of organ or tissue transplantation.

[0385] In some embodiments, cells of a particular cell type are collected from a patient or from a donor (e.g., from an HLA matched or mismatched donor who does not have a disease or disorder, for example), modified to express one or more (e.g., one, two, three, four, five, six, seven, or eight) masking transgenes, and then administered to a subject. This approach can be used to treat subjects carrying mutations in a particular gene, as the masked donor cells can endogenously express the wild-type version of the gene, or for subjects lacking a particular secreted protein or enzyme (e.g., using masked donor cells that endogenously express the protein or enzyme that is lacking in the subject). This approach can also be used to treat subjects receiving organ or tissue transplants, as the cells in the organ or tissue transplant can be modified to express one or more (e.g., one, two, three, four, five, six, seven, or eight) masking transgenes prior to transplantation.

[0386] Subjects that can be treated as described herein are subjects who have received a transplant, or subjects suffering from a disease or disorder described herein (e.g., wet AMD or retinal dystrophy, neurodegenerative diseases such as Parkinson's disease, myocardial infarction, osteoarthritis or RA, diabetes, hemophilia, metabolic disorders, or diseases or disorders listed in Table 2). The cells, compositions, and methods described herein can be used to treat diseases or disorders caused by or associated with cell loss, mutation or deficiency of a protein, or abnormal production of a protein, which can be treated using cell replacement proteins or cell therapy, the production of therapeutic proteins, the production of agonist antibodies, or the production of inhibitory antibodies. The methods described herein may include a step of screening a subject for mutations in genes associated with insufficient protein production before treating or administering a composition described herein. Standard methods known to those skilled in the art (e.g., genetic testing) can be used to screen for genetic mutations in a subject. The methods described herein may also include a step of evaluating the symptoms of a subject's disease or disorder before treating or administering a masked cell or composition described herein. After administration of the masked cells or compositions, the subject can be evaluated using the same diagnostic test to determine whether the subject's condition has improved. The compositions and methods described herein can be administered as a preventive treatment to a patient who has received a tissue or organ transplant before the patient exhibits any signs of tissue or organ rejection.

[0387] The masked cells, compositions, and methods described herein can be used to replace dead or dying cells in a subject (e.g., replacing neurons in a subject with a neurodegenerative disease, or replacing cardiomyocytes in a subject with a myocardial infarction). The masked cells, compositions, and methods described herein can also be used to provide immunosuppression in the area of ​​a tissue or organ transplant, or to reduce the risk of rejection of a tissue or organ transplant. Masked cells expressing a therapeutic agent (such as a protein or agonist antibody), compositions comprising such cells, or methods of administering such cells can be used to replace or provide a wild-type version of a protein that is mutated or absent in a subject (e.g., a protein that is produced but does not function properly due to a genetic mutation, such as a truncated protein or a protein with an altered charge, polarity, or binding pattern; or a protein that is not produced or is produced in insufficient amounts, such as insufficient protein production associated with a disease or disorder in Table 2). Masked cells expressing therapeutic agents (such as inhibitory or neutralizing antibodies), compositions comprising such cells, or methods of administering such cells can be used to block or neutralize proteins that are overexpressed or abnormally produced in a subject (e.g., proteins that are produced at a different time or location than the production of the protein in a healthy subject, such as abnormal protein production associated with a disease or disorder listed in Table 2).

[0388] Treatment may include administration of masked cells or compositions containing masked cells in various unit doses. Each unit dose will generally contain a predetermined amount of the masked cells described herein. The amount to be administered and the specific administration and formulation routes are within the skill of those skilled in the clinical field. The unit dose need not be administered as a single injection, but may include continuous infusion over a set period of time. Administration may be performed using a catheter, syringe, shunt, stent, microcatheter, pump, implanted with a device, or implanted with a stent. The number of cells administered may vary depending on whether the cells are administered to a tissue, organ, or body part associated with the disease or injury or subcutaneously to produce masked subcutaneous tissue. For administration to a tissue, organ, or body part, the masked cells may be, for example, 1 x 10 4 cells to 1x 10 10 cells (e.g., 1x 10 4 , 2x 10 4 , 3x10 4 , 4x 10 4 , 5x 10 4 , 6x 10 4 , 7x 10 4 , 8x 10 4 , 9x 10 4 , 1x 10 5 , 2x 10 5 , 3x 10 5 , 4x 10 5 , 5x 10 5 , 6x 10 5 , 7x 10 5 , 8x 10 5 , 9x 10 5 , 1x 10 6 , 2x 10 6 , 3x 10 6 , 4x 10 6 , 5x 10 6 , 6x 10 6 , 7x 10 6 , 8x10 6 , 9x 10 6 , 1x 10 7 , 2x 10 7 , 3x 10 7 , 4x 10 7 , 5x 10 7 , 6x 10 7 , 7x 10 7 , 8x 10 7 , 9x 10 7 , 1x 10 8 , 2x 108 , 3x 10 8 , 4x 10 8 , 5x 10 8 , 6x 10 8 , 7x 10 8 , 8x 10 8 , 9x 10 8 , 1x 10 9 , 2x 10 9 , 3x 10 9 , 4x10 9 , 5x 10 9 , 6x 10 9 , 7x 10 9 , 8x 10 9 , 9x 10 9 , 1x 10 10 The number of cells administered will depend on the size of the recipient tissue, organ, or body part. For example, 2.5 x 10 4 Up to 1x 10 5 cells (e.g., 2.5 x 10 4 , 3x 10 4 , 4x 10 4 , 5x 10 4 , 6x 10 4 , 7x 10 4 , 8x 10 4 , 9x 10 4 or 1x 10 5 cells) can be administered (e.g., injected) into the subretinal space of the eye or into specific brain regions; 1 x 10 6 Up to 1x10 8 cells (e.g., 1x 10 6 , 2x10 6 , 3x 10 6 , 4x 10 6 , 5x 10 6 , 6x 10 6 , 7x 10 6 , 8x 10 6 , 9x 10 6 , 1x 10 7 , 2x 10 7 , 3x 10 7 , 4x 10 7 , 5x 10 7 , 6x 10 7 , 7x 10 7 , 8x 10 7 , 9x 107 or 1x 10 8 cells) can be administered (e.g., injected) into the joint, where the amount of cells depends on the size of the joint; and 5 x 10 8 Up to 5x 10 9 cells (e.g., 5 x 10 8 , 6x 10 8 , 7x 10 8 , 8x10 8 , 9x 10 8 , 1x 10 9 , 2x 10 9 , 3x 10 9 , 4x 10 9 or 5x 10 9 To create a masked subcutaneous tissue, 8 x 10 cells can be administered subcutaneously (e.g., injected). 8 cells to 3x 10 9 cells (e.g., 8 x 10 8 , 9x 10 8 , 1x 10 9 , 2x 10 9 , 3x 10 9 The masked cells may be administered in two or more doses (e.g., two, three, four, five or more different doses, e.g., to different sized joints of a patient with RA) or in the same dose two or more times (e.g., two, three, four, five, six or more times over the course of an hour, a day, a week, a month, or a year). In some embodiments, the masked cells described herein are administered in the form of a tissue (e.g., a tissue that has been grown and / or differentiated in vitro from the masked cells). In some embodiments, the masked tissue is administered (e.g., implanted) with a gel, a biocompatible matrix, or a scaffold.

[0389] The compositions described herein are administered in an amount sufficient to prevent or reduce transplant rejection or to improve symptoms of a disease or condition listed in Table 2 (e.g., to reduce symptoms of osteoarthritis or RA (e.g., to reduce inflammation, joint pain, stiffness, or immobility); to reduce symptoms of retinal dystrophy or wet AMD (e.g., to improve vision, slow or reduce vascularization of the eye); to reduce symptoms of Parkinson's disease (e.g., to reduce tremor, rigidity, bradykinesia, or to improve posture or gait); to reduce symptoms of diabetes (e.g., to increase insulin levels, to reduce the need for regular insulin injections); to reduce symptoms of myocardial infarction (e.g., to improve cardiac function, to reduce infarct size); to reduce biological symptoms of hemophilia (e.g., to increase levels of clotting factors such as Factor VIII, to reduce excessive bleeding, to reduce bruising, to reduce nosebleeds, to reduce joint pain or swelling); or to reduce symptoms of a metabolic disorder (e.g., to increase appetite, growth or weight gain, or to reduce lethargy, weight loss, jaundice, seizures, abdominal pain, or vomiting)). Transplant rejection can be evaluated using standard methods known to those skilled in the art, and can be reduced by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) compared to transplant rejection rates typically observed without treatment. In some embodiments, administration of the masked cells or compositions described herein results in transplant rejection results equivalent to those observed in subjects administered immunosuppressants. Symptoms of the diseases and disorders described herein can be evaluated using standard methods known to those skilled in the art, and can be alleviated (e.g., the subject's condition can be improved) by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) compared to symptoms prior to administration of the masked cells or compositions described herein. These effects can occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks or longer after administration of the compositions described herein. Patients can be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer after administration of the masked cells or compositions, depending on the dosage and route of administration used for treatment. Depending on the results of the evaluation, the patient can receive additional treatment.

[0390] Combination therapy

[0391] In some embodiments, the masked cells described herein are administered in combination with one or more additional therapeutic agents. Additional therapeutic agents may be administered before, after, or simultaneously with the administration of the masked cells. Masked cells and additional therapeutic agents may also be administered simultaneously via co-formulation. Masked cells and therapeutic agents may also be administered sequentially so that the effects of the masked cells and therapeutic agents overlap, and their combined effect is to reduce symptoms or other parameters associated with the disorder greater than the reduction observed when the masked cells or therapeutic agents are delivered alone or in the absence of the other. The effects of the masked cells and therapeutic agents may be partially additive, fully additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of masked cells and therapeutic agents may be achieved by any appropriate route, including, but not limited to, oral, intravenous, intramuscular, topical, or subcutaneous routes. Masked cells and therapeutic agents may be administered by the same route or by different routes. For example, the masked cells may be administered by subcutaneous injection, while the additional therapeutic agent is administered orally. The masked cells can be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or up to 1-7, 1-14, 1-21, or 1-30 days before or after the additional therapeutic agent.

[0392] In one example, the additional therapeutic agent is one or more immunosuppressants commonly administered for organ or tissue transplantation. The immunosuppressant may be an agent administered immediately after transplantation to prevent acute rejection (e.g., methylprednisolone, atgam, antithymocyte globulin, OKT3, basiliximab or daclizumab) or an immunosuppressant for maintenance (e.g., prednisone), a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), mycophenolate mofetil, azathioprine or rapamycin). Other immunosuppressive agents administered after organ transplantation include corticosteroids (e.g., methylprednisolone, dexamethasone, prednisolone), cytotoxic immunosuppressants (e.g., azathioprine, chlorambucil, cyclophosphamide, mercaptopurine, methotrexate), immunosuppressant antibodies (e.g., antithymocyte globulin, basiliximab, infliximab), sirolimus derivatives (e.g., everolimus, sirolimus) and antiproliferative agents (e.g., mycophenolate mofetil, mycophenolate sodium and azathioprine). In this case, the masked cells are administered to or near the site of transplantation, or the tissue to be transplanted is modified to express one or more (e.g., one, two, three, four, five, six, seven or eight) masked transgenes, and immunosuppressants are administered as another source of immunosuppression when needed.

[0393] For use in treating inflammation and autoimmune-related diseases or disorders, the additional agent can be a disease-modifying antirheumatic drug (DMARD), a biologic response modifier (a type of DMARD), a corticosteroid, or a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, the additional agent is prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, or a biologic such as tofacitinib, adalimumab, abatacept, anakinra, anakinra, certolizumab pegol, etanercept, golimumab, infliximab, rituximab, or tocilizumab. In some embodiments, the additional agent is 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab (Lemtrada), aminosalicylate (5-aminosalicylic acid, sulfasalazine, mesalazine, balsalazide, olsalazine), antibiotics, antihistamines, anti-TNFα (infliximab, adalimumab, certolizumab pegol, natalizumab), azathioprine, belimumab, beta interferon, calcineurin inhibitors, certolizumab pegol, corticosteroids, cromolyn, cyclosporine A, cyclosporine, dimethyl fumarate (tecfidera), etanercept, gilentrol Gilenya, fumarate, Copaxone, golimumab, hydroxyurea, IFNγ, IL-11, infliximab, leflunomide, leukotriene receptor antagonists, long-acting beta2 agonists, mitoxantrone, mycophenolate mofetil, tysabri, ocrelizumab, pimecrolimus, probiotics (VSL#3), retinoids, rituximab, salicylic acid, short-acting beta2 agonists, sulfasalazine, tacrolimus, Aubagio, theophylline, tocilizumab, ustekinumab (anti-IL-12 / IL-23) or vedolizumab (anti-α3β7 integrin). In this case, the masked cells can be administered to replace tissues or organs damaged by inflammation or autoimmune-related diseases or disorders. In another example, the administered masked cells can be modified to express a biotherapeutic agent (e.g., an antibody) for the treatment of a specific inflammatory or autoimmune related disease or condition, and the additional agent can be a compound or a general anti-inflammatory agent (e.g., an NSAID or corticosteroid).

[0394] For example, if the disease is rheumatoid arthritis, the additional agent may be one or more of prednisone, prednisolone and methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide and azathioprine, tofacitinib, adalimumab, abatacept, anakinra, anakinra, certolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab. The cloaked cells administered may be cartilage or bone producing cells of the joint. In some embodiments, the cloaked cells may be modified to produce anti-TNFα antibodies and may be administered in combination with an anti-inflammatory agent (e.g., a corticosteroid).

[0395] In another example, for the treatment of AMD or retinal dystrophy, the additional therapeutic agent can be another biological agent (e.g., bevacizumab, ranibizumab or aflibercept), photodynamic therapy or photocoagulation. The cells of the shelter applied can be retinal cells (e.g., RPE cells). In some embodiments, the cells of the shelter can be modified to produce a VEGF inhibitor and can be administered in combination with photodynamic therapy or photocoagulation.

[0396] For use in the treatment of Parkinson's disease, the masked cells described herein can be administered with carbidopa-levodopa, dopamine agonists (e.g., pramipexole, ropinirole, rotigotine, or apomorphine), MAO-B inhibitors (e.g., selegiline, rasagiline), catechol-O-methyltransferase inhibitors (e.g., entacapone or tolcapone), anticholinergics (e.g., benztropine or trihexyphenidyl), amantadine, or deep brain stimulation. The administered masked cells can be dopaminergic neurons.

[0397] Additional agents used to treat myocardial infarction include anticoagulants (e.g., rivaroxaban, dabigatran, apixaban, heparin, warfarin), antiplatelet agents (e.g., aspirin, clopidogrel, dipyramidole, prasugrel, ticagrelor), angiotensin converting enzyme inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril), angiotensin II receptor blockers (e.g., candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan), angiotensin receptor neprilysin inhibitors (e.g., sacubitril / valsartan), beta blockers (e.g., acebutolol, atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, sotalol), combined alpha and beta blockers (e.g., carvedilol, labetalol hydrochloride), calcium Channel blockers (e.g., amlodipine, diltiazem, felodipine, nifedipine, nimodipine, nisoldipine, verapamil), cholesterol-lowering drugs (e.g., statins (e.g., atorvastatin, rosuvastatin), niacin (e.g., lovastatin), cholesterol absorption inhibitors (e.g., ezetimibe / simvastatin)), digitalis preparations (e.g., lanoxin), diuretics (e.g., amiloride, bumetanide, chlorothiazide, chlorthalidone, furosemide The occluded cells may be myocardial cells.

[0398] For use in treating infectious diseases, the additional agent can be an antiviral compound (e.g., vidarabine, acyclovir, ganciclovir, valganciclovir, a nucleoside analog reverse transcriptase inhibitor (NRTI) (e.g., AZT (zidovudine), ddI (didanosine), ddC (zalcitabine), d4T (stavudine) or 3TC (lamivudine)), a non-nucleoside reverse transcriptase inhibitor (NNRTI) (e.g., (nevirapine or delavirdine), a protease inhibitor (saquinavir, ritonavir, indinavir or nelfinavir), ribavirin or an interferon); an antibacterial compound; an antifungal compound; an antiparasitic compound. The administered masked cell can be an immune cell (e.g., a cell that can help fight infectious diseases, such as a masked T cell or B cell).

[0399] For use in treating diabetes, the additional agent may be insulin, a sulfonylurea (e.g., chlorpropamide, glipizide, glyburide, glimepiride), a biguanide (e.g., metformin), a meglitinide (e.g., repaglinide, nateglinide), a thiazolidinedione (e.g., rosiglitazone, pioglitazone), a DPP-4 inhibitor (sitagliptin, saxagliptin, linagliptin, alogliptin), a SGLT2 inhibitor (e.g., canagliflozin, dapagliflozin), an alpha-glucosidase inhibitor (e.g., acarbose, miglitol), a bile acid sequestrant (e.g., colesevelam), aspirin, or a dietary regimen. The administered masked cell may be a pancreatic beta cell, which may optionally be modified to express a transgene encoding insulin.

[0400] For use in treating hemophilia, the additional therapeutic agent may be a coagulation factor, desmopressin, a clot preserving drug (e.g., an antifibrinolytic agent such as aprotinin, aminocaproic acid, fibrinogen or tranexamic acid), a fibrin sealant or physical therapy. The occluded cells administered may be sinusoidal cells or endothelial cells, which may optionally be modified to express a transgene encoding Factor VIII.

[0401] For the treatment of metabolic deficiencies or disorders, the additional therapeutic agent may be a coenzyme (e.g., biotin, hydroxocobalamin, riboflavin, pyridoxine, folic acid, thiamine, ubiquinone, tetrahydrobiopterin), a bone marrow transplant, an organ transplant (e.g., a liver, kidney, or heart transplant), hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium chain triglycerides, miglustat, enzyme replacement therapy, or dietary restriction (e.g., a low protein or phenylalanine restricted diet for patients with phenylketonuria). The masked cell may be a cell carrying a wild-type copy of a gene that is mutated in a subject with a metabolic disorder, or a cell that endogenously produces an enzyme that is deficient in a subject with a metabolic disorder (e.g., a liver cell, a kidney cell, a heart cell, or any other cell that carries a wild-type copy of a gene that is mutated in a subject with a metabolic disorder or produces an enzyme that is deficient in a subject with a metabolic disorder).

[0402] For use in treating cancer, the additional agent can be a checkpoint inhibitor, a chemotherapeutic drug, a biological drug, a non-drug therapy (e.g., radiotherapy, cryotherapy, hyperthermia or surgical removal of tumor tissue), or an anti-cancer vaccine. The masked cells can be immune cells (e.g., macrophages, natural killer cells, dendritic cells, or T cells) that can help fight cancer.

[0403] Checkpoint inhibitors can be divided into at least 4 major categories: i) agents such as antibodies that directly block inhibitory pathways on T cells or natural killer (NK) cells (e.g., antibodies targeting PD-1 such as nivolumab, pidilizumab / CT-011, and pembrolizumab, antibodies targeting TIM-3, and antibodies targeting LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, or KIR), ii) agents such as antibodies that directly activate stimulatory pathways on T cells or NK cells (e.g., antibodies targeting OX40, GITR, or 4-1BB), iii) agents such as antibodies that block suppressive pathways on immune cells or rely on antibody-dependent cellular cytotoxicity to deplete suppressive populations of immune cells (e.g., antibodies targeting CTLA-4 such as ipilimumab or tremelimumab), antibodies targeting VISTA, and antibodies targeting PD-L2 (e.g., PDL2 / Ig fusion proteins such as AMP 224), Gr1 or Ly6G), and iv) agents such as antibodies or small molecules that directly block the inhibitory pathway on cancer cells or rely on antibody-dependent cellular cytotoxicity to enhance cytotoxic agents to cancer cells (e.g., rituximab, antibodies or small molecules targeting PD-L1 (e.g., MPDL3280A / RG7446; MEDI4736; MSB0010718C; BMS 936559) and antibodies or small molecule inhibitors targeting B7-H3 (e.g., MGA271), B7-H4, Gal-9 or MUC1). In one embodiment, the inhibitor of the checkpoint is an inhibitor of HVEM, CD160, CHK 1, CHK2, B-7 family ligands, or a combination thereof (e.g., an inhibitory antibody or small molecule inhibitor). Such agents described herein can be designed and produced, for example, by conventional methods known in the art (e.g., Templeton, Gene and Cell Therapy, 2015; Green and Sambrook, Molecular Cloning, 2012). In one embodiment, the inhibitor of the checkpoint is an inhibitory antibody (e.g., a monospecific antibody, such as a monoclonal antibody). The antibody can be, for example, humanized or fully human. In other embodiments, the inhibitor of the checkpoint is a fusion protein, such as an Fc-receptor fusion protein. In some embodiments, the inhibitor of the checkpoint is an agent that interacts with the checkpoint protein, such as an antibody. In other embodiments, the inhibitor of the checkpoint is an agent that interacts with the ligand of the checkpoint protein, such as an antibody.

[0404] Chemotherapeutic agents include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione substituted urea, methylhydrazine derivatives, adrenocortical suppressants, adrenocortical steroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. Also included are 5-fluorouracil (5-FU), folinic acid (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trihydroxymethylmelamine; acetylglucosamine (particularly bratacin and bratacinone); camptothecins (including the synthetic analog topotecan); bryostatin; spongestatin; CC-1065 (including its adolesine, carzeresine and biszeresine synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin tin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; mandarine; stolon; spongistatin; nitrogen mustards such as chlorambucil, naphthiazine, clofosamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nembixin, phenamicin, prednimustine, trofosamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g. calicheamicins, especially calicheamicin gamma II and calicheamicin omega II; danemycins, including danemycin A; bisphosphonates such as clodronate; esperamicins;and neocarcinogenin chromophores and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carrubicin, carminomycin, carmophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycin (such as mitomycin C), mycophenolic acid, noramycin, olivetomycin, peplomycin, porfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptomelanocin, streptozocin, tuberculocidin , ubenimex, netastatin, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethoate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as captestosterone, drostanolone propionate, cyclothiodine, melastosane, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostan; folic acid supplements such as folinic acid; acetoglucosalactone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; betribucil; bisantrene ; edatrexate; defofamin; colcemid; diazocone; eflornithine; elliptinium acetate; epicillon; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; nitraerine; pentostatin; methamine; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizolan; spirogermanium; tenuisporic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verrucosporin A, baculosporin A, and serpentin); urethane; vindesine; dacarbazine azine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; garcitocin; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; noantuo; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine;and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. Two or more chemotherapeutic agents may be used in a mixture for administration in combination with the masked cells described herein. Suitable dosing regimens for combination chemotherapy are known in the art. ;

[0405] Anticancer biologics include cytokines (e.g., interferons or interleukins (e.g., IL-2)) for cancer treatment. In other embodiments, biologics are anti-angiogenic agents, such as anti-VEGF agents, such as bevacizumab. In some embodiments, biologics are immunoglobulin-based biologics, such as humanized antibodies, fully human antibodies, Fc fusion proteins, or functional fragments thereof), which agonize targets to stimulate anticancer responses or antagonize antigens important to cancer. Such agents include rituximab; daclizumab; basiliximab; palivizumab; infliximab; trastuzumab; gemtuzumab ozogamicin; alemtuzumab; ibritumomab tiuxetan; adalimumab; omalizumab; tositumomab-I-131; efalizumab; cetuximab; bevacizumab; natalizumab; tocilizumab; panitumumab; ranibizumab; eculizumab; certolizumab pegol; golimumab; canakinumab; ustekinumab; ofatumumab; denosumab; motavizumab; recibazumab; belimumab; ipilimumab; brentuximab vedotin; pertuzumab; ado-trastuzumab emtacin; and obinutuzumab. Antibody-drug conjugates are also included.

[0406] Pill Box

[0407] The invention also features a kit containing a masked cell described herein (e.g., a masked cell expressing a set of masked transgenes described herein (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 of PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6), optionally further expressing one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1d39). In some embodiments, the masked cell is further modified to include one or more systems for regulating cell division (e.g., ALINK or EARC systems) and / or a therapeutic agent (e.g., a transgene encoding a protein or antibody). The masked cell can be provided in the form of a pharmaceutical composition. The kit may also include a syringe for administering the masked cells or pharmaceutical composition and instructions for administering the masked cells or pharmaceutical composition to treat a disease or condition described herein.

[0408] Example

[0409] The following examples are provided to further illustrate some embodiments of the present invention but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methods or techniques known to those skilled in the art may alternatively be used.

[0410] Example 1: Materials and Methods

[0411] Construction of vectors expressing target genes essential for allo-tolerance

[0412] Plasmids containing cDNA sequences of genes involved in allo-tolerance were obtained as follows:

[0413] PD-L1: Mount Sinai Hospital, clone #V102001

[0414] FasL: Mount Sinai Hospital, #75719

[0415] CD47: Mount Sinai Hospital, #V75535

[0416] CD200: GE Dharmacon, ID#17470

[0417] H2-M3: Mount Sinai Hospital, Clone #8188

[0418] Ccl21: Mount Sinai Hospital, clone #V77120

[0419] Mfge8: Mount Sinai Hospital, Clone #V72614

[0420] Spi6: Mount Sinai Hospital, clone #V8907

[0421] Expression vectors containing these target genes (GOI) or luciferase were generated using the Gateway cloning system (Thermo Fisher). Cd47, Ccl21, Mfge8, and Spi6 cDNAs were obtained with attB sites flanking the cDNA. For H2-M3, Cd200, FasL, and PD-L1, primers were designed to amplify the cDNA sequences and add attB sites ( Fig.15 (a)). After PCR amplification, the cDNA containing attB was recombined into the pDONR221 vector (Thermo Fisher, #1256017) by BP (recombination between attB and attP sites) reaction to generate the entry (pENTRY) clone ( Fig.15(b)). The BP reaction requires mixing the attB-flanked transgenic cDNA with the pDONR221 plasmid along with a buffer and BP enzyme provided by Invitrogen in a 1 mL test tube, where the BP enzyme recombines the GOI into the docking site of the pDONR221 plasmid. Insertion of the transgene into the pDONR221 plasmid was verified by DNA sequencing (TCAG sequencing facility at the Toronto Center for Applied Genomics). The pENTRY clone containing the GOI was then recombined into the destination vector via a gateway LR (recombination between attL and attR sites) reaction ( Fig.15 (c)). The LR reaction requires mixing the pDONR221 plasmid containing the GOI and the destination vector together with a buffer and LR enzyme provided by Invitrogen in a 1 mL tube, where the LR enzyme recombines the GOI cassette from the pDONR221 plasmid into the docking site of the destination plasmid. The destination vector used for all transgenic constructions contains a CAGG promoter followed by a Gateway entry site, an internal ribosome entry site (IRES), and a puromycin resistance selectable marker or a green fluorescent protein (GFP) reporter. The entire cassette is flanked by transposable PB sites. After LR recombination, the final destination vector containing the GOI was verified by restriction enzyme digestion ( Fig.15 (d)).

[0422] ES cell culture, transfection, selection and cloning

[0423] Mouse ES cells derived from the inbred C57BL / 6N mouse strain (Gertsenstein 2010) were cultured in DMEM high glucose supplemented with 15% fetal bovine serum (FBS, tested for compatibility with ES cell culture) and standard amounts of sodium pyruvate, non-essential amino acids (NEAA), Glutamax, penicillin / streptomycin, β-mercaptoethanol, and leukemia inhibitory factor (LIF) (Behringer et al. 2014). Cells were cultured on a feeder layer of mitomycin C-inactivated mouse embryonic fibroblasts (MEFs). Cultures were maintained in a standard cell culture incubator at 37°C and 5% CO2.

[0424] Transfections were performed using JetPRIME reagent (Polyplus, catalog number 14-07) following the manufacturer's protocol and were performed in three steps: 1) transfection with the PD-L1-IRES-GFP destination vector only, 2) transfection with all other transgenes carrying the puromycin selectable marker, and 3) transfection with the eLuciferase-IRES-GFP transgene.

[0425] Step 1: After transfection with PD-L1-IRES-GFP, cells were plated at low density so that after 5-6 days of multiple rounds of proliferation, single cell clones were selected based on the intensity of GFP expression - present as cell aggregates (colonies) and then expanded into clonal cell cultures. The clones with the highest and most consistent GFP expression were selected for the next step.

[0426] Step 2: After 24 hours of transfection with a transgenic containing a puromycin selection marker, puromycin was added to the culture medium. On the third day, cells were plated at clonal density and puromycin selection was continued until a single colony was picked and expanded into a clone. A large number of these clones were screened in vivo, and clones that were able to form teratomas in an allogeneic environment were designated as "NT2".

[0427] Step 3: NT2 was transfected with PB-CAG-eLuciferase-IRES-GFP as described above and plated at clonal density. GFP+ clones were picked and expanded. Ten clones with high levels of GFP expression were selected for further study.

[0428] Evaluation of transgene expression levels

[0429] RNA was isolated from cultures grown on 30 mm plates and from tumors grown in vivo. Cells were dissociated with trypsin, centrifuged and the supernatant removed. Cell pellets were immediately frozen on dry ice and stored at -80°C. Tumor tissue was dissected, immediately frozen on dry ice and stored at -80°C. RNA was isolated using the Sigma GeneElute Total RNA Miniprep Kit #RTN350 according to standard protocols. cDNA was obtained by reverse transcriptase reaction using the Qiagen Quantifast Reverse Transcriptase Kit #205313. Quantitative PCR was performed using Sensifast master mix from Bioline, #Bio-98020, gene-specific primers and 1:50 dilution of RNA. Samples were plated in 384-well plates using an Eppendorf epMotion 5070 robot and quantitative PCR was performed on a BioRad CFX384 Real-Time System C1000 thermal cycler according to standard protocols. qPCR data were captured by BioRad CFXManager 3.1 software and expression levels were calculated using Microsoft Excel.

[0430] Teratoma assay

[0431] Dilute Matrigel Matrix High Concentration (Corning catalog number 354248) 1:1 with cold DMEM medium on ice.7 The cells were suspended in 500uL of DMEM and an equal volume of Matrigel. 100ul of the suspension of 5x10 cells was subcutaneously injected into each dorsal flank of B6N (syngeneic) or FVB / N (allogeneic) mice. The resulting teratomas were formed 2-4 weeks after injection. The teratoma size was measured using a caliper and the volume was calculated using the formula V = (LxWxH) / 2. The tumor was allowed to grow to approximately 500mm 2 , which is a size that is well tolerated and also very suitable for downstream experiments. All transgenes are delivered into cells containing a "fail-safe system" (e.g., as described in WO 2016 / 141480, the entire contents of which are incorporated herein by reference. This genetic system allows complete inhibition of cell division by administration of ganciclovir (GCV). Once the teratomas from the aforementioned experiments reach 500 mm 2 , 50 mg / kg GCV was injected into the peritoneal cavity of mice every 2-3 days for 2-3 weeks. This treatment regimen resulted in an initial transient reduction in tumor size, which then stabilized at 400-500 mm after 2-3 weeks of treatment. 2 At the end of the experiment, mice were sacrificed and tumors were dissected. A small portion of the tissue was snap-frozen for RNA extraction, while the rest was fixed in 4% paraformaldehyde.

[0432] Bioluminescence imaging

[0433] Ten minutes before imaging, mice developing teratomas derived from cells transfected with the eLuciferase transgene were injected with 30 mg / mL of VivoGlo luciferin (Promega # P104C) at 100 uL / 25 g body weight. The animals were anesthetized with isoflurane and placed in an IVIS Lumina II imager (Caliper Life Sciences) driven by Living Image software. Depending on the signal intensity, the exposure time was set between 5 seconds and 5 minutes.

[0434] Histology

[0435] Fixed tumors were embedded in paraffin, sectioned and stained with hematoxylin / eosin for histological analysis at the CMHD pathology core. Histological images were processed with NDPview2 software.

[0436] Example 2: Generation of masked cells

[0437] Transgenes encoding the genes in Table 1 were cloned into expression vectors and sequence verified by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing, all using standard methods known in the art.

[0438] A set of constructs (Group 1) containing transgenes Cd47, Cd200, FasL and H2-M3 were transfected into mouse embryonic stem cells derived from the inventors' C57BL / 6 mouse ES line (C2). The presence of the transgenes was verified by PCR, and the expression of the expressed proteins was documented by immunohistochemistry ( Figure 1A -D). A second set of constructs (Set 2) containing the transgenes Ccl21, Mfge8, TGF-β and Spi6 were transfected into FVB / N derived ES cells (ES line C2).

[0439] A similar method was used to generate masked B16F10 melanoma cells except that the culture medium used was DMEM medium containing 10% fetal bovine serum (FBS).

[0440] Example 3: Screening method for inhibiting T cell activation

[0441] A modified in vitro mixed lymphocyte reaction (MLR) assay was used to screen transgene combinations that produced the most potent inhibition of T cell activation. Cell lines transfected with masking transgenes from Group 1 and Group 2 of Example 1 were used. Donor OT-1 splenocytes were labeled with carboxyfluorescein succinimidyl ester CFSE and 60,000 cells were added to each well of a 96-well plate. ES or melanoma cells were mixed 10:1 with oocyte expressing cells. 10,000 of these were added to each well of splenocytes. IL-2 was added as a general activator and T cell proliferation was measured by flow cytometry after 3 days ( Figure 2A-2E ). Cells were initially gated to include only CD8+ cells and all conditions were set up in quadruplicate.

[0442] Negative control (splenocytes only) produced a baseline proliferation rate of 6.12% ( Figure 2A Wild-type B16 melanoma (+10% oocyte expression) cells resulted in a significant acceleration of proliferation to 17.1% ( Figure 2B ), while masked cells reduced this proliferation to 9.51% ( Figure 2C ). and masked ES cells ( Figure 2E ) compared to the wild type ( Figure 2D ) obtained similar results.

[0443] Example 4: Studies using WT and masked cancer cells in syngeneic and allograft B16F10 melanoma cells

[0444] Since some candidate masking transgenes are intended to inhibit or modulate the initiation stages of the immune recognition cascade, the effects of these transgenes can be evaluated by MLR alone, as these events affect the maturation and physical migration of host APCs to local lymph nodes, where they subsequently activate naive T and B cells.

[0445] This requires an alternative assay that can screen a large number of transgene combinations in an in vivo allogeneic setting. Intraperitoneal and intravenous injections of ES cells carrying multiple transgene combinations have been attempted as a selection. However, teratoma formation depends on the aggregation of a minimum number of ES cells (1x10 5 – 5x10 6 , depending on the injection site), making this selection incompatible with this screen. However, the murine melanoma cancer cell line B16F10 derived from C57BL / 6 mice is not restricted in this manner. Intravenous injection of less than 5x10 3 This results in the effective induction of a large number of small cancer nodules in the lung. By limiting the number of cells injected, it is expected that cancer cells will be trapped in the alveoli and form nodules derived from a single or very small number of cells. By isolating these nodules and genotyping these nodules, the transgene can be identified.

[0446] Injection of B16F10 melanoma cells into the bloodstream of C57BL / 6 mice (isogenic graft control) resulted in the formation of cancer nodules in the lungs ( Figure 3A , left). However, the negative control - wild-type B16F10 melanoma transplanted into allogeneic control FVB mice - also developed small melanoma nodules in the lungs when observed at day 14 after injection. However, when the melanoma was allowed to grow for 24 days, the nodules almost completely regressed ( Figure 3A , right).

[0447] The above experiment was repeated by injecting a mixture of cancer cells expressing random combinations of candidate masking genes generated using the PiggyBac transposon system. Lung nodules that developed in the allogeneic setting contained one or more of the winning combinations required to protect the allograft from recognition and rejection ( Figure 3B , right). The same immune-masked cells also cause accelerated development in isogenic hosts ( Figure 3B , left picture).

[0448] Example 5: Non-cloaked embryonic stem cells do not form teratomas in an allogeneic environment

[0449] As shown in Table 4, it was confirmed that the wild-type ESC derived from C57BL / 6 mice could not form teratomas in FVB / N mice. Similarly, it was also shown that the wild-type ESC derived from FVB / N background could not form teratomas in C57BL / 6 hosts. ES cell colonies were dissociated with trypsin, washed once with DMEM without additives, and resuspended in Matrigel HC at a concentration of about 50 million cells per milliliter. Recipient mice were anesthetized and injected subcutaneously with one hundred microliters in each flank region. The teratomas in development were tracked for 12 weeks and verified by palpation and measuring the volume with a caliper.

[0450] Table 4: Teratomas formed in FVB / N mice and C57BL / 6 hosts (injected with wild-type ESCs derived from C57BL / 6 mice or wild-type ESCs derived from FVB / N background)

[0451]

[0452] Example 6: Masked ES cells can be propagated in both syngeneic and allogeneic hosts

[0453] To verify the cloaking ability of candidate transgenes, ESCs were transfected with the same transgenes, while also adding a luciferase transgene that can be detected by imaging. Briefly, ES cells were prepared as described above. The presence of live cells was measured repeatedly by imaging. Figure 4 The images in were taken on day 17 after injection.

[0454] exist Figure 4 The upper panel shows the proliferation of immune-masked cells in a syngeneic host, while the lower panel shows the proliferation of immune-masked cells in an allogeneic host.

[0455] In another experiment, masked ES cells from C57BL / 6 mice with high expression of 8 immunomodulatory transgenes (clone NT2) were injected subcutaneously into different allogeneic mouse strains (C3H, FVB / N, and CD1) with mismatched MHC alleles. The red arrow indicates the teratoma formed ( Figures 5A-5C ).

[0456] Example 7: Mice with masked tissues are not immune compromised

[0457] Non-immune-masked (wild-type) ESCs are transplanted into mice carrying existing immune-masked tissue and the mice are evaluated to determine whether they can effectively reject the non-immune-masked graft ( Figure 6 ). The same mice were imaged several times over a 15-day period. Figure 6 As shown in the left figure, in the syngeneic mouse control, the graft was not rejected over time. Figure 6 The left mouse in the right image has a pre-existing immune-masked graft (arrow). Figure 6 The middle mouse in the right panel of had been previously implanted with C57BL / 6 allogeneic ESCs but rejected the transplant (although not limited to theory, rejection may be due to preformed antibodies against C57BL / 6 cells). Figure 6The mouse on the right in the right picture of had never been transplanted before. All three mice successfully rejected the non-immune masked graft. The mouse on the right was slower to reject the graft, probably because it did not have any pre-formed antibodies against the C57BL / 6 cells.

[0458] Similar experiments were performed in which wild-type embryonic stem cells were detected until 9 days after injection into FVB / N mice with concealed teratomas ( Figure 7 ). However, at day 12, no evidence of remaining cells could be detected. Control animals were C57BL / 6 mice that also carried obscured tumors. The signal in these mice increased over the course of the experiment.

[0459] Example 8: Masked and fail-safe embryonic stem cell lines

[0460] When a fail-safe C57BL / 6 ES cell line (e.g., as described in WO / 2016 / 141480) was co-transfected with five candidate masking transgenes (PD-L1, FasL, Cd47, Cd200, and H2-M3), none of these transgenic lines produced teratomas in an allograft setting. When the set of co-transfected genes was amplified by three additional candidate masking genes: Spi6, Ccl21b, and Mfge8, 38 clonal lines were generated. One of these lines, NT2, produced teratomas in an allogeneic recipient (FVB). The 38 clonal lines (including the NT2 line) were measured using quantitative PCR (see Figures 8A-8H The expression level of the masked gene in Figures 8A-8H ). Among the 38 clones, NT2 was the highest overexpressor of Ccl21b (16,000x), FasL (25,000x), Cd200 (1700x), Cd47 (16x), Mfge8 (34x), Spi6 (600x), and H2-M3 (750x) compared to WT ES cells. PD-L1, although not the highest level expressor among the clones, was also significantly increased at 350x relative to ES cells. The expression of these genes was also examined in the Project Grandiose dataset (www.stemformatics.org / project_grandiose), and it was found that Ccl21b, FasL, Cd200, PD-L1, and Spi6 expression were below the detection threshold, so their expression relative to ES cells was very high. Based on this data, these eight highly activated genes may have a major role in inducing immune tolerance in allografts.

[0461] NT2 cells were injected into C57BL / 6 to form FVB allogeneic cells ( Figures 11A-11BTeratomas were generated in the allogeneic environment of FVB isoC57Bl / 6. From day 12 to day 38, allogeneic teratomas (n=6) grew stably. 2 At a size of 1.5 × 10 mM, ganciclovir (GCV) treatment begins to remove the proliferative components of the tumor ( Figures 12A-12B , the above figure ( Fig. 12A ) isogenic teratoma; bottom image ( Fig. 12B ) allogeneic teratomas). 20 days of treatment terminated allograft growth. This experiment shows that: 1) fail-safe and masked (NT2) cell-derived teratomas respond similarly to GCV treatment; they enter dormancy after brief GCV exposure; 2) teratomas remain stable after GCV. There are no signs of rejection of dormant tissue; and 3) the kinetics of teratoma growth in FVB animals are different from C57BL / 6.

[0462] Masked transgenes expressed at high levels survive to form teratomas in allogeneic mice. In our system, masked transgenes are expressed under a very strong synthetic promoter, CAG ( Fig.19 ). The CAG promoter is a combination of the cytomegalovirus early enhancer element, the spliceosomal acceptor of the rabbit β-globin gene, and also the promoter, first exon, and first intron of the chicken β-actin gene. Extensive qPCR analysis of the levels of transgene transcripts in many different ES cell clones was performed, each with different transgene expression levels. Only those ES clones with the highest expression of the masked transgene were able to survive in the allogeneic host.

[0463] like Fig. 9 As shown, the transcript expression level of the immunomodulatory gene relevant to the masking technique changes between ES cell clones. Concentric circles are depicted with log10 scale. The black thick line is 1x, and the next outer ring is 10x, and then 100x. The innermost ring is 0.1x. All values ​​are normalized to the positive control, which is the activated leukocytes separated from the mouse lymphoid organs, and its natural expression immunomodulatory transgene. The upper left figure shows that there is no transgenic modified wild-type ES cell for reference-they express little or no relevant immunomodulatory transgene. In contrast, clone NT...

Claims

1. A cell genetically modified to contain a set of transgenes comprising: PD-L1, HLA-G, Cd47, Cd200, FASLG, Ccl21, Mfge8 and Serpin B9.

2. A cell genetically modified to contain a set of transgenes comprising: PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8 and Spi6.

3. The cell of claim 1 or 2, further comprising one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1d39, or a gene encoding a biologic that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1d39.

4. The cell of claim 3, wherein the TGF-β or biologic acts locally in the transplant environment.

5. The cell of claim 1 or 2, wherein the cell is a cell suitable for genome editing.

6. The cell of claim 1 or 2, wherein the cell is a source of a therapeutic cell type.

7. The cell of claim 1 or 2, wherein the cell is an induced pluripotent stem cell.

8. The cell of claim 1 or 2, wherein the cell is a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem cell or progenitor cell, a germline stem cell, a lung stem cell or progenitor cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, an intestinal stem cell or progenitor cell, an amniotic stem cell, an umbilical cord blood stem cell, or a neural stem cell or progenitor cell.

9. The cell of claim 1 or 2, wherein the cell is an adult stem cell.

10. The cell of claim 1 or 2, wherein the cell is a tissue-specific stem cell or a totipotent stem cell.

11. The cell of claim 5 or 6, wherein the cell is a fibroblast, a monocyte precursor, a B cell, an exocrine cell, a pancreatic progenitor cell, an endocrine progenitor cell, a hepatoblast, a myoblast, a preadipocyte, a hepatocyte, a smooth muscle cell, a K562 human erythroleukemia cell line, a bone cell, a synoviocyte, a tendon cell, a ligament cell, a meniscus cell, an adipocyte, a dendritic cell, a natural killer cell, a skeletal muscle cell, a cardiomyocyte, an erythroid-megakaryocyte, an eosinophil, a macrophage, a T cell, a pancreatic beta cell, a neuron, an exocrine progenitor cell, a duct cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a PP cell, a bile duct cell, an epidermal keratinocyte, an epithelial cell, a kidney cell, a skeletal joint synovial cell, a periosteal cell, an endothelial cell, a pericardial cell, a meningeal cell, a keratinocyte precursor cell, a keratinocyte stem cell, a pericyte, a glial cell, an ependymal cell, a cell isolated from an amniotic or placental membrane, or a serosal cell.

12. The cell of claim 5 or 6, wherein the cell is a somatic cell.

13. The cell of claim 5 or 6, wherein the cell is derived from the skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach.

14. The cell of claim 5 or 6, wherein the cell is a chondrocyte, a blood cell, a white or brown fat cell, a hormone-secreting cell, or a perichondrial cell.

15. The cell of claim 1 or 2, wherein the cell further comprises at least one mechanism for controlling cell proliferation, the genetically modified cell comprising: A genetic modification of one or more cell division loci / locus (CDL), which is one or more endogenous or exogenous loci whose one or more transcription products are expressed by dividing cells, wherein the genetic modification is one or more of the following: i) an abrogation ligation (ALINK) system comprising a DNA sequence encoding a negative selectable marker transcriptionally linked to a DNA sequence encoding the CDL; and ii) An exogenous activator-regulated CDL (EARC) system comprising an inducible activator-based gene expression system operably linked to the CDL.

16. The cell of claim 15, wherein the genetic modification of the CDL comprises targeted replacement of the CDL with one or more of: a) a DNA vector comprising the ALINK system; b) a DNA vector comprising the EARC system; and c) a DNA vector comprising the ALINK system and the EARC system; Wherein the ALINK system and / or the EARC system are each operably connected to the CDL.

17. The cell of claim 15, wherein the genetic modification of the CDL comprising the ALINK system is homozygous, heterozygous, hemizygous or compound heterozygous, and / or the genetic modification of the CDL comprising the EARC system leads to activation of the CDL only by the inducer of the gene expression system based on the inducible activator.

18. The cell of claim 15, wherein the CDL is one, two or more of the following loci: i)Actr8, Alg11, Anapc11, Anapc2, Anapc4, Anapc5, Aurka, Banf1, Birc5, B ub3, Casc5, Ccna2, Ccnh, Cdc123, Cdc16, Cdc20, Cdc23, Cdk1, Cenpa, Cenpm Chek1, Chmp2a, Ckap5, Cltc, Cops5, Dctn2, Dctn3, Dhfr, Dtl, Dync1h1, Ec d, Ect2, Ep300, Ercc3, Espl1, Fntb, Gadd45gip1, Gins1, Gnb2l1, Gspt1, Hau s1, Haus3, Haus5, Haus8, Hdac3, Kif11, Kif23, Kpnb1, Mastl, Mau2, Mcm3, M cm4, Mcm7, Mnat1, Mybbp1a, Ncapd2, Ncaph, Ndc80, Nle1, Nsl1, Nudc, Nuf2, N up133, Nup160, Nup188, Nup214, Nup85, Orc3, Pafah1b1, Pcid2, Pfas, Phb2 Pkmyt1, Plk1, Pmf1, Pole2, Ppat, Psma6, Psma7, Psmb1, Psmb4, Psmd12, Psm d13, Psmd14, Psmd7, Racgap1, Rad21, Rae1, Rcc1, Rfc3, Rps27a, Rrm2, Sae1 Sec13, Smarcb1, Smc2, Smc4, Son, Spc24, Spc25, Terf2, Tpx2, Tubg1, Tubgc p2, Tubgcp5, Tubgcp6, Txnl4a, Usp39, Wdr43, Zfp830, Aatf, Alyref, Brf2. Cdc45, Cdc6, Cdt1, Cinp, Cirh1a, Ddb1, Ercc2, Gabpb1, Gtf2b, Gtf2h4, Gtf3 a, Gtf3c1, Gtf3c2, Hinfp, Ints3, Kin, Mcm2, Mcm6, Mcrs1, Med11, Mtpap, My c, Ndnl2, Nol11, Nol8, Pcna, Pola1, Pold2, Pole, Polr1a, Polr3a, Polr3c, P olr3h, Prmt1, Prmt5, Puf60, Rad51, Rad51c, Rbx1, Rfc2, Rfc4, Rfc5, Rpa1. Rps3, Rrm1, Ruvbl1, Ruvbl2, Sap30bp, Smc1a, Smc3, Snapc4, Snapc5, Snip1<h2 style=";text-align:left;direction:ltr">Srrt、Ssrp1、Taf10、Taf1c、Taf6、Taf6l、Ticrr、Top1、Top2a、Trrap、Zbtb1 1、Actl6a、Atr、Chd4、Ciao1、Ddx21、Dnaja3、Dnmt1、Gins2、Gtf2h3、Mms22l、 Mtor、Narfl、Ndufa13、Nol12、Nup107、Oraov1、Pam16、Pola2、Ppie、Prpf19 、Psmc5、Rbbp5、Rbbp6、Rptor、Rrn3、Smg1、Supt6、Tada2b、Tfip11、Tonsl、Tp t1、Uba1、Vps25、Wbscr22、Wdr5、Xab2、Zmat2、Zfp335、Acly、Adsl、Ahcy、Ar l2、Chka、Coasy、Cox4i1、Ddx10、Ddx20、Dhdds、Dhx30、Dhx8、Dhx9、Dlst、Dpa gt1、Gfpt1、Gmps、Gpn1、Gpn3、Guk1、Hsd17b10、Lrr1、Mtg2、Myh9、Nampt、Nc bp1、Nfs1、Ppcdc、Qrsl1、Rpp14、Smarca4、Snrnp200、Srbd1、Srcap、Ube2i、U be2m、Vcp、Aamp、Acin1、Aco2、Adss、Alg2、Ap2s1、Arcn1、Armc7、Atp2a2、At p5a1、Atp5d、Atp5o、Atp6v0b、Atp6v0c、Atp6v1a、Atp6v1b2、Atp6v1d、Aurka ip1、Ccdc84、Cct2、Cct3、Cct4、Cct5、Cct7、Cct8、Cdipt、Cenpi、Chordc1、C oa5、Cog4、Copa、Copb1、Copb2、Cope、Copz1、Coq4、Cox15、Cox17、Cse1l、Csn k2b, Cycs, Dad1, Dap3, Dctn5, Ddost, Dgcr8, Dhodh, Dnlz, Dnm1l, Dnm2, Dohh, Dolk, Donson, Dph3, Dtymk, Eif2b2, Eif2s2, Emc1, Emc7, Eno1, Fam50a, Fa m96b、Fdps、Gapdh、Gart、Gemin4、Gemin5、Ggps1、Gmppb、Gnb1l、Gosr2、Gpk ow、Gpn2、Gps1、Grpel1、Grwd1、Hmgcr、Hmgcs1、Hspa5、Hspa9、Hspd1、Hspe1、<h2 style=";text-align:left;direction:ltr">Hyou1、Ipo13、Iscu、Itpk1、Kansl2、Kansl3、Kri1、Lamtor2、Leng8、Ltv1、M ak16、Mat2a、Mcm3ap、Mdn1、Mmgt1、Mrpl16、Mrpl17、Mrpl33、Mrpl38、Mrpl3 9、Mrpl45、Mrpl46、Mrpl53、Mrps22、Mrps25、Mrps35、Mrps5、Mvd、Mvk、Naa2 5、Napa、Nat10、Ndor1、Ndufab1、Nol10、Nop9、Nrde2、Nsf、Nubp1、Nudcd3、Nu p155、Nup93、Nus1、Nvl、Ogdh、Osbp、Pak1ip1、Pfdn2、Pgam1、Pkm、Pmpcb、Pp il2、Ppp4c、Prelid1、Prpf31、Prpf6、Psma1、Psma2、Psma3、Psmb2、Psmb3、Ps mb5、Psmb6、Psmb7、Psmc2、Psmc3、Psmc4、Psmd1、Psmd2、Psmd3、Psmd4、Psmd 6、Psmg3、Ptpmt1、Ptpn23、Rabggta、Rabggtb、Rbm19、Rfk、Rheb、Riok1、Rpn1 、Rtfdc1、Sacm1l、Samm50、Sco2、Sdha、Sdhb、Sec61a1、Slc20a1、Slc7a6os、 Smn1、Smu1、Snrpd1、Snrpd3、Snrpe、Spata5、Spata5l1、Tango6、Tbcb、Tbcc 、Tbcd、Tcp1、Telo2、Tex10、Tfrc、Timm10、Timm13、Timm23、Timm44、Tmx2、T npo3、Trmt112、Trnau1ap、Ttc1、Ttc27、Tti1、Tti2、Txn2、Uqcrc1、Uqcrh、Ur b2、Vmp1、Vps29、Vps52、Wars2、Wdr7、Wdr70、Wdr74、Wdr77、Yae1d1、Yrdc、Z nhit2、Aars、Bms1、Bud31、Bysl、Cars、Cdc5l、Cdc73、Cebpz、Clasrp、Clp1、C ox5b、Cpsf1、Cpsf2、Cpsf3l、Dars、Dbr1、Ddx18、Ddx23、Ddx24、Ddx41、Ddx4 6、Ddx47、Ddx49、Ddx54、Ddx56、Dgcr14、Dhx15、Dhx16、Dhx38、Diexf、Dimt1、Dis3、Dkc1、Dnajc17、Ears2、Ebna1bp2、Eef1a1、Eef1g、Eef2、Eftud2、Eif1 ad、Eif2b1、Eif2b3、Eif2s1、Eif3c、Eif3d、Eif3f、Eif3g、Eif3i、Eif3l、Ei f4a1、Eif4a3、Eif4g1、Eif5b、Eif6、Elac2、Ell、Etf1、Exosc2、Exosc4、Exo sc5、Exosc7、Exosc8、Fars2、Farsa、Farsb、Fau、Fip1l1、Ftsj3、Gle1、Gnl3l 、Gtf2e1、Gtpbp4、Hars、Hars2、Heatr1、Hnrnpc、Hnrnpk、Hnrnpl、Hnrnpu、I ars、Iars2、Imp3、Imp4、Ints1、Ints4、Ints5、Ints8、Ints9、Isg20l2、Kars 、Lars、Lars2、Las1l、Lrpprc、Lsm2、Lsm3、Lsm7、Magoh、Mars、Mars2、Med17 、Med20、Med22、Med27、Med30、Med8、Mepce、Mettl16、Mphosph10、Mrpl10、Mr pl12、Mrpl21、Mrpl28、Mrpl3、Mrpl34、Mrpl4、Mrpl41、Mrpl51、Mrps14、Mrp s15、Mrps16、Mrps18a、Mrps2、Mrps21、Mrps24、Mrps6、Nars、Nars2、Ncbp2、 Nedd8, Ngdn, Nhp2, Nip7, Noc2l, Noc4l, Nol6, Nol9, Nop16, Nop2, Nop58, Ns a2、Nudt21、Osgep、Pabpn1、Pdcd11、Pes1、Phb、Phf5a、Pnn、Polr1b、Polr1c、 Polr2a、Polr2b、Polr2c、Polr2d、Polr2f、Polr2g、Polr2h、Polr2i、Polr2j 、Polr2l、Polr3e、Pop1、Pop4、Ppa1、Ppan、Ppp2ca、Prim1、Prpf38b、Prpf4、 Prpf8, Ptcd1, Pwp2, Qars, Ran, Rars, Rars2, Rbm25, Rbm8a, Rbmx, Rcl1, Rng tt、Rnmt、Rnpc3、Rpap1、Rpl10、Rpl10a、Rpl11、Rpl12、Rpl13、Rpl14、Rpl15、Rpl18, Rpl18a, Rpl23, Rpl24, Rpl26, Rpl27, Rpl27a, Rpl3, Rpl30, Rpl31, Rpl32, Rpl35, Rpl35a, Rpl36, Rpl37, Rpl37a, Rpl38, Rpl4, Rpl5, Rpl6, Rpl7, Rpl7a, Rpl7l1, Rpl8, Rpl9, Rplp0, Rpp21, Rpp30, Rps10, Rps11, Rps12, Rps13, Rps15, Rps15a, Rps16, Rps17, Rps19, Rps2, Rps21, Rps23, Rps25, Rps4x, Rps5, Rps6, Rps7, Rps8, Rps9, Rpsa, Rsl24d1, Sars, Sars2, Sart1, Sart3, Sdad1, Sf1, Sf3a1, Sf3a2, Sf3a3, Sf3b2, Sf3b3, Sf3b4, Sfpq, Sin3a, Smg5, Smg6, Snrnp25, Snrnp27, Snrpd2, Snrpf, Srrm1, Srsf1, Srsf2, Srsf3, Srsf7, Ssu72, Sugp1, Tars, Tars2, Tbl3, Thoc2, Thoc5, Thoc7, Timeless, Tsen2, Tsr1, Tsr2, Tufm, Tut1, Twistnb, U2af1, U2af2, Uba52, Ubl5, Upf1, Upf2, Utp15, Utp20, Utp23, Utp3, Utp6, Vars, Wars, Wdr12, Wdr3, Wdr33, Wdr36, Wdr46, Wdr61, Wdr75, Xpo1, Yars, Yars2, Ythdc1, Zbtb8os and Zc3h3; or, ii)ACTR8, ALG11, ANAPC11, ANAPC2, ANAPC4, ANAPC5, AURKA, BANF1, BIRC5 BUB3, CASC5, CCNA2, CCNH, CDC123, CDC16, CDC20, CDC23, CDK1, CENPA, CENPM CHEK1, CHMP2A, CKAP5, CLTC, COPS5, DCTN2, DCTN3, DHFR, DTL, DYNC1H1, EC D, ECT2, EP300, ERCC3, ESPL1, FNTB, GADD45GIP1, GINS1, GNB2L1, GSPT1, HAU S1, HAUS3, HAUS5, HAUS8, HDAC3, KIF11, KIF23, KPNB1, MASTL, MAU2, MCM3, M CM4, MCM7, MNAT1, MYBBP1A, NCAPD2, NCAPH, NDC80, NLE1, NSL1, NUDC, NUF2, N UP133, NUP160, NUP188, NUP214, NUP62, NUP85, ORC3, PAFAH1B1, PCID2, PFA S, PHB2, PKMYT1, PLK1, PMF1, POLE2, PPAT, PSMA6, PSMA7, PSMB1, PSMB4, PSMD 12. PSMD13, PSMD14, PSMD7, RACGAP1, RAD21, RAE1, RCC1, RFC3, RPS27A, RRM 2 SAE1, SEC13, SMARCB1, SMC2, SMC4, SON, SPC24, SPC25, TERF2, TPX2, TUBG1 TUBGCP2, TUBGCP5, TUBGCP6, TXNL4A, USP39, WDR43, ZNF830, AATF, ALYREF BRF2, CDC45, CDC6, CDT1, CINP, CIRH1A, DDB1, ERCC2, GABPB1, GTF2B, GTF2H 4. GTF3A, GTF3C1, GTF3C2, HINFP, HIST2H2AA3, INTS3, KIN, MCM2, MCM6, MCR S1, MED11, MTPAP, MYC, NDNL2, NOL11, NOL8, PCNA, POLA1, POLD2, POLE, POLR1 A, POLR2J2, POLR3A, POLR3C, POLR3H, PRMT1, PRMT5, PUF60, RAD51, RAD51C. RBX1, RFC2, RFC4, RFC5, RPA1, RPS3, RRM1, RUVBL1, RUVBL2, SAP30BP, SMC1A.<h2 style=";text-align:left;direction:ltr">SMC3、SNAPC4、SNAPC5、SNIP1、SRRT、SSRP1、TAF10、TAF1C、TAF6、TAF6L、TIC RR、TOP1、TOP2A、TRRAP、ZBTB11、ACTL6A、ATR、CHD4、CIAO1、DDX21、DNAJA3、 DNMT1、GINS2、GTF2H3、HIST2H2BF、MMS22L、MTOR、NARFL、NDUFA13、NOL12、N UP107、ORAOV1、PAM16、POLA2、PPIE、PRPF19、PSMC5、RBBP5、RBBP6、RPTOR、R RN3, SMG1, SUPT6H, TADA2B, TFIP11, TONSL, TPT1, UBA1, VPS25, WBSCR22, WDR5, XAB2, ZMAT2, ZNF335, ACLY, ADSL, AHCY, ARL2, CHKA, COASY, COX4I1, COX 7C、CTPS1、DDX10、DDX20、DHDDS、DHX30、DHX8、DHX9、DLST、DPAGT1、GFPT1、G MPS、GPN1、GPN3、GUK1、HSD17B10、LRR1、MTG2、MYH9、NAMPT、NCBP1、NFS1、PPC DC、QRSL1、RPP14、SMARCA4、SNRNP200、SRBD1、SRCAP、UBE2I、UBE2M、VCP、AA MP、ACIN1、ACO2、ADSS、ALG2、AP2S1、ARCN1、ARMC7、ATP2A2、ATP5A1、ATP5D、 ATP5O、ATP6V0B、ATP6V0C、ATP6V1A、ATP6V1B2、ATP6V1D、AURKAIP1、C1orf1 09、C21orf59、CCDC84、CCT2、CCT3、CCT4、CCT5、CCT7、CCT8、CDIPT、CENPI、C HORDC1、COA5、COG4、COPA、COPB1、COPB2、COPE、COPZ1、COQ4、COX15、COX17、 CSE1L、CSNK2B、CYCS、DAD1、DAP3、DCTN5、DDOST、DGCR8、DHODH、DNLZ、DNM1L 、DNM2、DOHH、DOLK、DONSON、DPH3、DTYMK、EIF2B2、EIF2S2、EMC1、EMC7、ENO1 、FAM50A、FAM96B、FDPS、GAPDH、GART、GEMIN4、GEMIN5、GGPS1、GMPPB、GNB1L、<h2 style=";text-align:left;direction:ltr">GOLGA6L1、GOSR2、GPKOW、GPN2、GPS1、GRPEL1、GRWD1、HMGCR、HMGCS1、HSPA5 、HSPA9、HSPD1、HSPE1、HYOU1、IPO13、ISCU、ITPK1、KANSL2、KANSL3、KRI1、L AMTOR2、LENG8、LTV1、MAK16、MAT2A、MCM3AP、MDN1、MFAP1、MMGT1、MRPL16、M RPL17、MRPL33、MRPL38、MRPL39、MRPL45、MRPL46、MRPL53、MRPS22、MRPS25、M RPS35、MRPS5、MVD、MVK、NAA25、NAPA、NAT10、NDOR1、NDUFAB1、NOL10、NOP9、 NRDE2、NSF、NUBP1、NUDCD3、NUP155、NUP93、NUS1、NVL、OGDH、OSBP、PAK1IP1、 PFDN2、PGAM1、PKM、PMPCB、PPIL2、PPP4C、PRELID1、PRPF31、PRPF6、PSMA1、P SMA2、PSMA3、PSMB2、PSMB3、PSMB5、PSMB6、PSMB7、PSMC2、PSMC3、PSMC4、PSMD 1、PSMD2、PSMD3、PSMD4、PSMD6、PSMG3、PTPMT1、PTPN23、RABGGTA、RABGGT、 RBM19、RFK、RHEB、RIOK1、RPN1、RTFDC1、SACM1L、SAMM50、SCO2、SDHA、SDHB、 SEC61A1、SLC20A1、SLC7A6OS、SMN1、SMU1、SNRPD1、SNRPD3、SNRPE、SPATA5、 SPATA5L1、TANGO6、TBC1D3B、TBC1D3C、TBCB、TBCC、TBCD、TCP1、TELO2、TEX10 、TFRC、TIMM10、TIMM13、TIMM23、TIMM44、TMX2、TNPO3、TRMT112、TRNAU1AP、 TTC1、TTC27、TTI1、TTI2、TUBB、TXN2、UQCRC1、UQCRH、URB2、VMP1、VPS28、VPS 29、VPS52、WARS2、WDR7、WDR70、WDR74、WDR77、YAE1D1、YRDC、ZNHIT2、AARS、 BMS1、BUD31、BYSL、CARS、CDC5L、CDC73、CEBPZ、CLASRP、CLP1、COX5B、CPSF1、<h2 style=";text-align:left;direction:ltr">CPSF2、CPSF3L、DARS、DBR1、DDX18、DDX23、DDX24、DDX41、DDX46、DDX47、DDX 49、DDX54、DDX56、DGCR14、DHX15、DHX16、DHX38、DIEXF、DIMT1、DIS3、DKC1、 DNAJC17、EARS2、EBNA1BP2、EEF1A1、EEF1G、EEF2、EFTUD2、EIF1AD、EIF2B1、 EIF2B3、EIF2S1、EIF3C、EIF3CL、EIF3D、EIF3F、EIF3G、EIF3I、EIF3L、EIF4A 1、EIF4A3、EIF4G1、EIF5B、EIF6、ELAC2、ELL、ETF1、EXOSC2、EXOSC4、EXOSC5 、EXOSC6、EXOSC7、EXOSC8、FARS2、FARSA、FARSB、FAU、FIP1L1、FTSJ3、GLE1、 GNL3L、GTF2E1、GTPBP4、HARS、HARS2、HEATR1、HNRNPC、HNRNPK、HNRNPL、HNR NPU、IARS、IARS2、IMP3、IMP4、INTS1、INTS4、INTS5、INTS8、INTS9、ISG20L2、 KARS、KIAA0391、LARS、LARS2、LAS1L、LRPPRC、LSM2、LSM3、LSM7、MAGOH、MAR S、MARS2、MED17、MED20、MED22、MED27、MED30、MED8、MEPCE、METTL16、MPHOS PH10、MRPL10、MRPL12、MRPL21、MRPL28、MRPL3、MRPL34、MRPL4、MRPL41、MRP L51、MRPS14、MRPS15、MRPS16、MRPS18A、MRPS2、MRPS21、MRPS24、MRPS6、NAR S、NARS2、NCBP2、NEDD8、NGDN、NHP2、NIP7、NOC2L、NOC4L、NOL6、NOL9、NOP16 、NOP2、NOP58、NSA2、NUDT21、OSGEP、PABPN1、PDCD11、PES1、PHB、PHF5A、PNN 、POLR1B、POLR1C、POLR2A、POLR2B、POLR2C、POLR2D、POLR2F、POLR2G、POLR2 H、POLR2I、POLR2J、POLR2L、POLR3E、POP1、POP4、PPA1、PPAN、PPP2CA、PRIM1、PRPF38B, PRPF4, PRPF8, PTCD1, PWP2, QARS, RAN, RARS, RARS2, RBM25, RBM8A, RBMX, RCL1, RNGTT, RNMT, RNPC3, RPAP1, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL14, RPL15, RPL18, RPL18A, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL3, RPL30, RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL37, RPL37A, RPL38, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL7L1, RPL8, RPL9, RPLP0, RPP21, RPP30, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS19, RPS2, RPS21, RPS23, RPS25, RPS3A, RPS4X, RPS5, RPS6, RPS7, RPS8, RPS9, RPSA, RSL24D1, SARS, SARS2, SART1, SART3, SDAD1, SF1, SF3A1, SF3A2, SF3A3, SF3B2, SF3B3, SF3B4, SFPQ, SIN3A, SMG5, SMG6, SNRNP25, SNRNP27, SNRPD2, SNRPF, SRRM1, SRSF1, SRSF2, SRSF3, SRSF7, SSU72, SUGP1, TARS, TARS2, TBL3, THOC2, THOC5, THOC7, TIMELESS, TSEN2, TSR1, TSR2, TUFM, TUT1, TWISTNB, U2AF1, U2AF2, UBA52, UBL5, UPF1, UPF2, UTP15, UTP20, UTP23, UTP3, UTP6, VARS, WARS, WDR12, WDR3, WDR33, WDR36, WDR46, WDR61, WDR75, XPO1, YARS, YARS2, YTHDC1, ZBTB8OS and ZC3H3., 19. The cell of claim 18, wherein the CDL encodes a gene product that functions in one or more of the following: cell cycle, DNA replication, RNA transcription, protein translation, and metabolism.

20. The cell of claim 19, wherein the CDL is one, two or more of CDK1, TOP2A, CENPA, BIRC5, and EEF2.

21. The cell of claim 20, wherein the CDL is CDK1.

22. The cell of claim 15, wherein the ALINK system comprises a herpes simplex virus-thymidine kinase / ganciclovir system, a cytosine deaminase / 5-fluorocytosine system, a carboxylesterase / irinotecan system, or an iCasp9 / AP1903 system.

23. The cell of claim 22, wherein the ALINK system is the herpes simplex virus-thymidine kinase / ganciclovir system.

24. The cell of claim 15, wherein the EARC system is a dox-bridge system, a coumarate switch inducible system, an ecdysone inducible system, a radio wave inducible system, or a ligand reversible dimerization system.

25. The cell of claim 24, wherein the ERAC system is a dox-bridge system.

26. The cell of claim 1 or 2, wherein all eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 are expressed at a level greater than the expression level of the corresponding endogenous genes in the cell or equal to or greater than the expression level of the corresponding endogenous genes in activated leukocytes.

27. The cell of claim 1 or 2, wherein: a) the PD-L1 transgene encodes a protein consisting of the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12; b) the HLA-G or H2-M3 transgene encodes a protein consisting of the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 15; c) the Cd47 transgene encodes a protein consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; and / or d) The CD200 transgene encodes a protein consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

28. The cell of claim 3, wherein the IFNγR1 d39 transgene encodes a protein consisting of the amino acid sequence of SEQ ID NO:

17.

29. The cell of claim 1 or 2, wherein each of the transgenes is operably linked to a constitutive promoter.

30. The cell of claim 29, wherein the constitutive promoter is selected from the group consisting of a CAG promoter, a cytomegalovirus (CMV) promoter, an EF1α promoter, a PGK promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of Moloney virus, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

31. The cell of claim 1 or 2, wherein the cell further comprises a transgene encoding a therapeutic agent.

32. The cell of claim 31, wherein the therapeutic agent is a protein.

33. The cell of claim 31, wherein the therapeutic agent is an antibody.

34. The cell of claim 33, wherein the antibody is an inhibitory antibody or an agonist antibody.

35. The cell of claim 31, wherein the therapeutic agent is administered by: a) expressed by an inducible expression system selected from the group consisting of a tetracycline response element, a light-inducible system, a radiogenetic system, a coumarate switch-inducible system, an ecdysone-inducible system, a destabilizing domain system, or a ligand-reversible dimerization system; or b) expressed by a constitutive promoter selected from the group consisting of: CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, Epstein-Barr virus (EBV) promoter and Rous sarcoma virus (RSV) promoter.

36. The cell of claim 1 or 2, wherein all eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 are expressed at a level within the top 5% of gene expression of all genes in the genome of the cell.

37. A genetically modified cell population comprising the cells of claim 1 or 2.

38. A composition comprising the cell of any one of claims 1 to 36 or the cell population of claim 37.

39. The composition of claim 38, further comprising a pharmaceutically acceptable excipient.

40. A kit comprising the composition of claim 38.

41. Use of the composition of claim 38 in the preparation of a medicament for treating a disease or disorder in a subject in need thereof.

42. The method of claim 41, wherein the disease or disorder is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, retinal dystrophy, infectious disease, or myocardial infarction.

43. The use of claim 41, wherein the disease or disorder is a metabolic disorder, an autoimmune disease, age-related macular degeneration, or hemophilia.

44. The use of claim 41, wherein the cell or cell population is differentiated into a lineage-restricted cell type.

45. The use according to claim 44, wherein: a) the disease or disorder is myocardial infarction, and the cell or cell population is differentiated into cardiomyocytes; b) the disease or disorder is blindness, and the cell or cell population differentiates into a photoreceptor cell; c) the disease or disorder is spinal cord injury, Parkinson's disease, Huntington's disease or Alzheimer's disease, and the cell or cell population is differentiated into neurons; or d) the disease or disorder is multiple sclerosis, and the cell or cell population is differentiated into glial cells.

46. ​​The use of claim 41, wherein the medicament is formulated for local administration to a tissue or body part in need of cells or the therapeutic agent.

47. The use of claim 41, wherein the medicament is formulated for administration intravenously, subcutaneously, intramuscularly, transdermally, intradermally, parenterally, intraarterially, intravascularly, or by infusion.

48. The use of claim 47, wherein the medicament is formulated for administration by subcutaneous injection to produce a concealed subcutaneous tissue.

49. The use of claim 41, wherein the medicament is formulated for administration to a tissue.

50. The use of claim 49, wherein the drug is formulated for administration with a gel, a biocompatible matrix or a cell scaffold.

51. The use of claim 41, wherein the medicament comprises 25,000 to 5,000,000,000 cells.

52. The use of claim 47, wherein the medicament comprises 800,000,000 to 3,000,000,000 cells.

53. The use of claim 41, wherein the medicament is formulated for administration with an additional therapeutic agent.

54. The use according to claim 53, wherein: a) the additional therapeutic agent is for administration prior to administration of the medicament; b) the additional therapeutic agent is for administration after administration of the medicament; or c) the additional therapeutic agent is for administration simultaneously with the administration of the medicament.

55. The use of claim 53, wherein the additional therapeutic agent is an immunosuppressant, a disease-modifying antirheumatic drug (DMARD), a corticosteroid or a nonsteroidal anti-inflammatory drug (NSAID), prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, tofacitinib, adalimumab, abatacept, anakinra, anakinra, certolizumab, etanercept, golimumab, infliximab, rituximab or tocilizumab, 6-mercaptopurine, 6-thioguanine, alemtuzumab , aminosalicylates, antibiotics, antihistamines, anti-TNFα, azathioprine, belimumab, interferon beta, calcineurin inhibitors, corticosteroids, comaline, cyclosporine A, cyclosporine, dimethyl fumarate, fingolimod, fumarate esters, copaxone, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonists, long-acting beta2 agonists, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, probiotics, retinoids, salicylic acid, short-acting beta2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, ustekinumab Mab or vedolizumab, bevacizumab, ranibizumab or aflibercept, carbidopa-levodopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase inhibitors, anticholinergics, amantadine, anticoagulants, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, angiotensin receptor neprilysin inhibitors, beta blockers, combined alpha and beta blockers, calcium channel blockers, cholesterol-lowering drugs, niacin, cholesterol absorption inhibitors, digitalis preparations, diuretics, vasodilators, antiviral compounds, nucleoside analog reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), protease inhibitor, antibacterial compound, antifungal compound, antiparasitic compound, insulin, sulfonylurea, biguanide, meglitinide, thiazolidinedione, DPP-4 inhibitor, SGLT2 inhibitor, alpha-glucosidase inhibitor, bile acid sequestrant, aspirin, coagulation factor, desmopressin, clot preserving drug, fibrin sealant, coenzyme, bone marrow transplant, organ transplant, medium chain triglyceride, miglustat, checkpoint inhibitor, chemotherapeutic drug, biologic drug, hyperthermia, or anticancer vaccine.

56. The use of claim 41, wherein the use comprises controlling the proliferation of the cell or cell population.

57. The method of claim 56, wherein the cell or cell population comprises an ALINK system, and controlling proliferation comprises: i) allowing proliferation of said cell or cell population comprising said ALINK system by maintaining said cell or cell population comprising said ALINK system in the absence of an inducer of said negative selectable marker; or ii) eliminating or inhibiting the proliferation of said cells or cell populations comprising said ALINK system by exposing said cells or cell populations comprising said ALINK system to an inducer of said negative selectable marker.

58. The method of claim 56, wherein the cell or cell population comprises an EARC system, and controlling cell proliferation comprises: i) allowing proliferation of said cells or cell population comprising said EARC system by exposing said cells or cell population comprising said EARC system to an inducer of said inducible activator-based gene expression system; or ii) preventing or inhibiting the proliferation of said cell or cell population comprising said EARC system by maintaining said cell or cell population comprising said EARC system in the absence of an inducer of said inducible activator-based gene expression system.

59. The use of claim 41, wherein the use comprises removing the cell or cell population after completion of the therapy.

60. Use of the composition of claim 38 in the preparation of a medicament for providing local immunosuppression at a transplantation site in an allogeneic host.

61. The use according to claim 60, wherein: a) the allogeneic host is a mammal; and / or b) The allogeneic host suffers from a degenerative disease or condition treatable by cell therapy.

62. The use according to claim 61, wherein: a) the allogeneic host is a mouse or a human; and / or b) the disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, retinal dystrophy, infectious disease, myocardial infarction or a degenerative disease.

63. The use of claim 61, wherein the disease or disorder is an enzyme or hormone deficiency or an age-related disease.

64. The use of claim 61, wherein the disease or disorder is a metabolic disorder, an autoimmune disease, age-related macular degeneration, or hemophilia.

65. The use of claim 60, wherein the use further comprises controlling proliferation of the cell or cell population at a transplantation site in the allogeneic host, wherein: (a) the cell or cell population comprises an ALINK system, and controlling the proliferation of the cell or cell population comprises: i) allowing proliferation of said cell or cell population comprising said ALINK system by maintaining said cell or cell population comprising said ALINK system in the absence of an inducer of said negative selectable marker; or ii) eliminating or inhibiting the proliferation of said cell or cell population comprising said ALINK system by exposing said cell or cell population comprising said ALINK system to an inducer of said negative selectable marker; and / or (b) the cell or cell population comprises the EARC system, and controlling the proliferation of the cell or cell population comprises: i) allowing proliferation of said cells or cell population comprising said EARC system by exposing said cells or cell population comprising said EARC system to an inducer of said inducible activator-based gene expression system; or ii) preventing or inhibiting the proliferation of said cell or cell population comprising said EARC system by maintaining said cell or cell population comprising said EARC system in the absence of an inducer of said inducible activator-based gene expression system.

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