Allograft tolerance without the need for systemic immune suppression

Abstract

To provide a cell genetically modified to provide a local immunosuppression at a transplant site when transplanted in an allogeneic host, and to provide methods for making and using the same.SOLUTION: Provided is a cell comprising a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting, and whose function is one or more of: mitigating antigen presenting cell activation and function; mitigating graft attacking leukocyte activity or cytolytic function; mitigating macrophage cytolytic function and phagocytosis of allograft cells; inducing apoptosis in graft attacking leukocytes; mitigating local inflammatory proteins; and protecting against leukocyte-mediated apoptosis.SELECTED DRAWING: Figure 1A-D

Description

[Technical Field]

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

[0002] The emergence 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 within the human body. These properties have enabled researchers to better understand human development and the pathogenesis of developmental disorders. Researchers have also given modern medicine powerful new tools for treating diseases that have been difficult or impossible to treat with conventional medicine, including spinal cord injury, diabetes, blindness, multiple sclerosis, and cancer, to name a few. The effectiveness and range of diseases amenable to cell therapy will only increase as our understanding of how stem cell differentiation is controlled and the biology of differentiated cell products increases.

[0003] These applications present significant and significant challenges. In addition to cell safety, one of the most important concerns is immune rejection of cells from different genetic backgrounds. Immune rejection remains a significant barrier because the immune system has evolved a complex set of mechanisms to recognize and eliminate "non-self" cells that express specific protein fragments (particularly those derived from the major histocompatibility complex (MHC) in mice and HLA in humans)) that differ between donors and recipients (Yang et al., Nat Rev Genet. 18:309-26 (2017)). This response is almost certainly a by-product of evolutionary pressure to protect against opportunistic infections and malignancies (often defined by the presence of "foreign" proteins and epitopes). Depending on the context, rejection of transplanted cells or tissues can occur over time scales of minutes / hours (hyperacute), days / months (acute), and months / years (chronic) (LaRosa et al., J Immunol. 178:7503-9 (2007)). This rejection results from the complex and coordinated effects of cell types derived 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)).

[0004] One of the most important pathways for rejection is the priming 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 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 the transplanted cells or tissues and kill them, accompanied by the release of cytolytic factors such as perforin and granzymes. NK cells can also induce apoptosis in donor cells based on foreign or absent 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 with the release of proinflammatory cytokines at the transplant site (Mannon, Curr Opin Organ Transplant. 17:20-5 (2012)). Many other cell types and subtypes also play a role in allograft rejection. These, along with allograft rejection, are highly conserved across vertebrate species because of the same immune pathways used to eliminate common viral and bacterial pathogens.

[0005] Current solutions to prevent allograft rejection include two options: finding a donor with a matched histocompatibility haplotype (most likely from a genetically related family) and, more commonly, using broadly directed immunosuppressive drugs (Wiseman, Clin J Am Soc Nephrol. 11:332-43 (2016); Malaise et al., Transplant Proc. 37:2840-2 (2005)). Common medications include those from the families of 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 medications must be taken daily for life, and even a missed dose can increase the risk of rejection. However, they do not always work, and when they do, rates of chronic rejection still continually rise over time (Demetris et al., Ann Transplant. 2:27-44 (1997); Libby et al., Immunity. 14:387-97 (2001)). Most importantly, they are systemically acting, ultimately rendering patients immunocompromised with increased rates of cancer and life-threatening infections (Gallagher et al., J Am Soc Nephrol. 21:852-8 (2010)).In the context of ES cells, these drugs have shown only modest improvement in enabling survival across MHC barriers (Swijnenburg et al., Proc Natl Acad Sci US A. 105:12991-6 (2008); Torimi et al., Neurol Res. 31:220-7 (2009)). Newer, more targeted immunosuppressants are becoming available and are being tested in the skin and heart (Larsen et al., Nature. 381:434-8 (1996)) and in ES cell allograft settings (Pearl et al., Cell Stem Cell. 8:309-17 (2011)), but they remain systemically acting and therefore likely leave host immunity compromised.

[0006] One proposed advantage of iPS cell discovery was that they could be generated from and for each patient. These cells, in theory, should be protected from immune rejection by the corresponding patient (Pearl et al., Sci Transl Med. 4:164ps25 (2012)). However, induction of the iPS cell state involves epigenetic changes and in vitro culture pressures that can result in abnormalities and malignancies, and therefore each cell line must be rigorously tested and / or genetically modified to achieve safety and functionality (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 generate and test iPS cell lines for each individual patient makes this approach practical and economically impractical. Even if costs were dramatically reduced, it would not be able to help patients who require immediate treatment for conditions such as burns, heart attacks, strokes, and spinal cord injuries (among others). Furthermore, given recent findings, it is debatable whether iPS cell-derived cell types are truly protected from immune rejection, even when transplanted into the same host from which they were derived (Zhao et al., Nature. 474:212-5 (2011)).

[0007] One proposed solution in this regard has been the use of naturally suppressive or regulatory immune cells, such as Tregs, that are expanded and / or transplanted 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 are suggested based on the recognition of suppressive immune pathways, particularly the discovery of the master regulator FoxP3, which programs a subset of CD4+ regulatory T cells (Hori et al., 299:1057-61(2003); Fontenot et al., Nat Immunol. 4:330-6(2003)), and the demonstration of their critical importance in promoting tolerance to allografts (Kendal et al., J Exp Med. 208:2043-53(2011)). This idea contrasts with some of the first tolerance induction strategies, which focused almost exclusively on depletion of effector T cells with monoclonal antibodies combined with bone marrow transplantation and the creation 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 suppressor T cell phenotype was later appreciated with strategies that blocked key T cell receptors in a way that did not kill the cells but left them unresponsive to the allograft (Cobbold et al., J Immunol. 172:6003-10 (2004)), yet could simultaneously suppress naive T cells of other specificities (Cobbold et al., Immunol Rev. 129:165-201 (1992); Qin et al., Eur J Immunol. 20:2737-45 (1990)).These cells, now recognized as Tregs, express 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. al., Nature. 450:566-9 (2007)) and preferential consumption 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)), as well as local ATP (Regateiro et al., Eur J Immunol. 41:2955-65 (2011); Regateiro et al., Clin Exp Immunol. 171:1-7 (2013)) or essential amino acids (Cobbold et al., Proc Natl Acad Sci US Tolerance may be promoted by a number of mechanisms, including, but not limited to, depletion of IL-1, IL-2, and IL-1 (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-36, IL-37, IL-38, IL-49, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-4

[0008] Two approaches for the potential therapeutic use of Tregs include either in vitro expansion using donor antigens coupled with transplantation or selective in vivo expansion that exploits differences between regulatory and effector T cells. While these strategies are intriguing, to date, long-term allograft acceptance has not been demonstrated solely through the use of in vitro or in vivo expanded Tregs. Many complications and unknown aspects of Treg biology remain, including optimal methodologies for in vitro or in vivo expansion, as well as the associated dosage and timing of treatment. Furthermore, antigen-specific Treg suppression can be "overcome" depending on the inflammatory context (Korn et al., Nat Med. 13:423-31 (2007)), and Tregs have been shown to be killed by NK cells (Roy et al., J Immunol. 180:1729-36 (2008)).

[0009] In addition to Tregs, other suppressor cell types, such as antigen-presenting cells (DCs), are also being explored to induce allograft tolerance (Walker et al., Trends Immunol. 36:63-70 (2015)). DCs are the link between innate and adaptive immunity and can induce both effector and suppressor immune responses depending on their maturation state and context, such as local inflammatory cues. During allograft rejection, DCs present allograft antigens on their surface within the binding groove of MHC (mouse) or HLA (human) molecules, along with costimulatory molecules such as CD80, CD86, and CD40 (among others), which are recognized and activated by allograft-specific T cell clones (Walker et al., Trends Immunol. 36:63-70 (2015)). Tolerogenic DCs can be induced from an immature state by exposure to suppressive cues that maintain low levels of expression of MHC and costimulatory molecules and then promote naive T cells to anergic or even T regulatory subtypes upon DC-T cell interaction.

[0010] Therapeutically, one application of this biology is to expand DCs that are simultaneously exposed in vitro to specific allograft antigens of interest 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, and drugs such as corticosteroids, cyclosporine, tacrolimus, rapamycin, aspirin, mecophenolate mofetil, sanglifehrin, and deoxyspergualin (Hackstein et al., Nat Rev Immunol. 4:24-34 (2004)). Alternatively, DCs have been genetically engineered to directly express immune regulators such as TGF-β, IL-10, VEGF, FasL, CTLA4-Ig, IDO, NFKb decoy receptor, soluble TNFR, CCR7, and siRNA-induced silencing of IL-12 (Morelli et al., Immunol Rev. 196:125-46 (2003)). These cultured or engineered DCs are then transferred into recipients simultaneously with the allograft to test whether they can prolong allograft survival by suppressing allograft-specific T cells or increasing the number of allograft-localized T regulatory cells.

[0011] In one prototype approach of this kind, bone marrow-derived DCs were transduced with SOCS1 (preventing upregulation of costimulatory molecules and MHCII), which prolonged mouse cardiac allografts (Fu et al., Cell Mol Immunol. 6:87-95 (2009)). In another demonstration, FasL-expressing DCs were also able to prolong mouse cardiac allografts (Min et al., J Immunol. 164:161-7 (2000)). In general, there are many specific and combinatorial approaches using tolerogenic DCs along these lines (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)), with highly variable results depending on the type of DC modification, culture conditions, timing, and type of allograft tested (Zhou et al., J Immunol 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 trials have begun, including a safety study 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)).

[0012] Many unknowns remain regarding both adoptive Treg and tolerogenic DC therapies, one of the most important being the duration of their effectiveness. In vivo studies have shown that prolonged allograft survival is possible using these two approaches (with or without additional immunosuppressants), but not for long periods of time; in almost all cases, the allograft ultimately dies. This is consistent with the fact that both Tregs and DCs have a finite time span. It is also possible to "convert" and instead promote inflammatory pathways among tolerogenic phenotypes, particularly DCs (Delamarre et al., Semin Immunol. 23:2-11 (2011); Schreibelt et al., Cancer Immunol Immunother. 59:1573-82 (2010); Satpathy et al., Nat Immunol. 14:937-48 (2013)). This is likely due to the highly adaptive nature of DCs and their ability to sense and respond to a wide range of inflammatory cues. These cells have also been shown to be able to die very rapidly after adoptive transfer in vivo. Also, many subsets of suppressive Tregs and tolerogenic DCs have been described, and it remains unclear which subtype is the ideal subtype or whether it depends entirely on the context of allograft transplantation.

[0013] Furthermore, these types of approaches pose significant practical and economic barriers in that they require clinicians to manipulate and work with complex immune cell types in addition to the therapeutic ones. Given their finite lifespan, it remains unclear whether these cells will need to be delivered continuously and / or repeatedly to confer long-term tolerance to allografts. This complicates the already expensive and time-consuming methodologies for culturing, expanding, or transducing cells with important immune modulators, ultimately preventing their use for extremely time-sensitive treatments.

[0014] Another approach to inducing tolerance is the use of hematopoietic cell transplantation (HCT), in which the recipient of an HLA-mismatched organ undergoes 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 results in chimerism, which may allow 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 hematopoietic cells play in positive and negative selection in the thymus, where they eliminate cells with affinity for hematopoietic cell-containing antigens that may also be present in the allograft, ultimately leading to their rejection (Griesemer et al., Transplantation. 90:465-74 (2010)). However, an inherent and dangerous risk of this approach is the possibility of graft-versus-host disease (GVHD), in which transplanted hematopoietic cells recognize recipient tissue as foreign and systemically attack it (Sun et al., PLoS One. 7:e52096 (2012)). Since its inception, several variants of HCT have been developed to suppress rejection, including the use of non-myeloablative strategies.These strategies use modified chemotherapy regimens, often with lower dosages, so that recipients undergoing HCT do not undergo complete ablation of their hematopoietic compartment. For example, one recent example of these strategies used tolerance-promoting cell (FC)-based HCT to promote tolerance in HLA-mismatched kidney recipients while largely avoiding GHVD (Leventhal et al., Sci Transl Med. 4:124ra28 (2012)).

[0015] In addition to the risks of GHVD and secondary HCT procedures, a common limitation to these chimerism-inducing approaches is the need to have an HLA-matched donor available for bone marrow collection. This is easily achieved in rodent studies but is highly demanding in humans. The donor organ should ideally be harvested from the donor as soon as possible, leaving an incredibly short window for bone marrow collection, if possible. It is also an expensive and logistically demanding procedure requiring a highly patient- and surgery-specific approach. Furthermore, as with the regulatory cell approach, it is unclear how this approach would be practically applicable in situations where patients would benefit from treatment for acute injury or disease or require immediate treatment.

[0016] Another approach tested to reduce allorejection in vivo is the removal of histocompatibility molecules (Torikai et al., Blood. 122:1341-9 (2013)), which are the primary antigenic source of "non-self" recognition in allorejection. This is consistent with empirical data showing that HLA-matched donors and recipients significantly improve organ survival after transplantation (Opelz et al., Rev Immunogenet. 1:334-42 (1999)). While this approach has had some positive results, removal of MHC class I makes cells highly 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)). This approach 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 address differences in antigens outside the MHC / HLA gene family (minor antigens) (Roopenian et al., Immunol Rev. 190:86-94 (2002)).

[0017] This approach involved the deletion of all classical HLA class I molecules from pluripotent stem cells in combination with the introduction of a gene encoding HLA-E, the minimally polymorphic HLA that inhibits NK cells (Gornalusse et al., Nat Biotechnol. (2017)). This approach demonstrated short-term resistance to NK and CD8 T cell attack in partially immunodeficient humanized mice, but it was not demonstrated that these cells could survive long-term in a fully immunocompetent host. In another approach, ES cells were engineered to express PD-L1 and CTLA4-Ig, which improved survival in allogeneic hosts (Rong et al., Cell Stem Cell. 14:121-30 (2014)), but with the severe limitation that CTLA4-Ig can lead to systemic immunosuppression. It remains to be demonstrated that a series of modifications to ES or iPS cells enables them to escape allogeneic rejection without the potential for systemic immunosuppression and the need for immunosuppressive drugs. Summary of the Invention [Problem to be solved by the invention]

[0018] It is an object of the present disclosure to mitigate and / or eliminate one or more of the above disadvantages. [Means for solving the problem]

[0019] In one aspect, cells are provided that are genetically modified to contain at least one mechanism for providing local immunosuppression at the transplant site when transplanted into an allogeneic host. The genetically modified cells comprise a set of transgenes, each transgene encoding a cytoplasmic, membrane-bound, or locally acting gene product having one or more of the following functions: a) attenuating antigen-presenting cell activation and function, b) attenuating graft-attacking leukocyte activity or cytolytic function, c) attenuating macrophage cytolytic function and phagocytosis of allograft cells, d) inducing apoptosis in graft-attacking leukocytes, e) attenuating local inflammatory proteins, and f) protecting against leukocyte-mediated apoptosis.

[0020] In an 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 version of HLA-G, H2-M3), Cd47, Cd200, FASLG (or the mouse version of FASLG, FasL), Ccl21 (or the mouse version of Ccl21, Ccl21b), Mfge8, and Serpin B9 (or the mouse version of Serpin B9, Spi6).

[0021] In an 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).

[0022] In a cellular embodiment, the set of transgene genes includes genes encoding PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8, and Serpin B9(Spi6), or a biological substance that acts as an agonist of PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8, and Serpin B9(Spi6).

[0023] In embodiments 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 genes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or genes encoding a biological substance that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.

[0024] In cellular embodiments, the TGF-β or biological agent acts locally within the graft environment. In cellular embodiments, the TGF-β or biological agent acts locally within the graft environment with minimal systemic effects.

[0025] In various embodiments of the cell, the cell is a stem cell, a cell amenable to genome editing, and / or a source of a therapeutic cell type (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 cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germline stem cells, pulmonary stem cells or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte progenitor cells, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, osteocytes, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, nasal cells, and the like. The cell types include neural killer cells, skeletal muscle cells, cardiac myocytes, erythroid-megakaryocyte cells, eosinophils, macrophages, T cells, islet 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 adipocytes, hormone-secreting cells, epidermal keratinocytes, epithelial cells, kidney cells, germ cells, skeletal joint synoviocytes, periosteal cells, perichondrocytes, 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, serous cells, somatic cells, or cells derived from the skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.

[0026] In embodiments 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., reducing the tumorigenicity of the modified cell or reducing the proliferation of modified cells that have become tumorigenic). The genetically modified cell includes a genetic modification of one or more (e.g., one, two, three, or more) cell division loci (CDLs), where a CDL is one or more loci whose transcription products are expressed by dividing cells (e.g., all dividing cells that contain one or more of the immunosuppressive transgenes), and the genetic modification is one or more of: a) an ALINK system, which includes a DNA sequence encoding a negative selection marker transcriptionally linked to a DNA sequence encoding the CDL, and b) an exogenous activator of regulation of a CDL (EARC) system, which includes an inducible activator-based gene expression system operably linked to the CDL.

[0027] In a cellular embodiment, genetic modification of the CDL comprises targeted replacement of the CDL using one or more of: a) a DNA vector comprising an ALINK system; b) a DNA vector comprising an EARC system; and c) a DNA vector comprising an ALINK system and an EARC system, wherein the ALINK and / or EARC system are each operably linked to the CDL.

[0028] 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 through the EARC-modified allele.

[0029] 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 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.

[0030] 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.

[0031] In various embodiments of the cell, the EARC system is a doxycycline-induced "dox-crosslinking" system, a coumate switch-induced system, an ecdysone-induced system, a radio-wave-induced system, or a ligand-reversible dimerization system; preferably, the EARC system is a dox-crosslinking system.

[0032] In one aspect, a method is provided for providing local immunosuppression at a transplant site in an allogeneic host, the method comprising providing cells and expressing a set of transgenes in the cells, each transgene encoding a cytoplasmic, membrane-bound, or locally acting gene product having one or more of the following functions: a) attenuating antigen-presenting cell activation and function, b) attenuating graft-attacking leukocyte activity or cytolytic function, c) attenuating macrophage cytolytic function and phagocytosis of allograft cells, d) inducing apoptosis in graft-attacking leukocytes, e) attenuating local inflammatory proteins, and f) protecting against leukocyte-mediated apoptosis.

[0033] In an embodiment of this 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 genes encoding a biological agent that acts as an agonist of PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8, or Serpin B9(Spi6).

[0034] In an embodiment of this 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 Serpin B9 (Spi6).

[0035] In an embodiment of this method, the set of transgene genes includes genes encoding PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8, and Serpin B9(Spi6), or a biological substance that acts as an agonist of PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8, and Serpin B9(Spi6).

[0036] In embodiments 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 genes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or genes encoding a biological agent that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39. In embodiments of the method, the TGF-β or biological agent acts locally within the graft environment. In embodiments, the TGF-β or biological agent acts locally within the graft environment with minimal systemic effects.

[0037] In various embodiments of the methods, the cells are stem cells, cells amenable to genome editing and / or a source of a therapeutic cell type (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 cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germline stem cells, pulmonary stem cells or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte progenitor cells, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, osteocytes, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, nasal cells, and the like. The cell types include neural killer cells, skeletal muscle cells, cardiac myocytes, erythroid-megakaryocyte cells, eosinophils, macrophages, T cells, islet 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 adipocytes, hormone-secreting cells, epidermal keratinocytes, epithelial cells, kidney cells, germ cells, skeletal joint synoviocytes, periosteal cells, perichondrocytes, 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, serous cells, somatic cells, or cells derived from the skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.

[0038] In various embodiments of the method, the cells are provided (e.g., injected) at or near the transplant site. In various embodiments of the method, the cells are provided (e.g., injected or transplanted) into a graft (e.g., injected or transplanted into a tissue or organ graft before, during, or after transplant). In some embodiments, the cells in which the transgene is expressed are cells of the graft (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)).

[0039] In one aspect, a method of controlling proliferation of cells at a transplant site in an allogeneic host is provided (e.g., to reduce the tumorigenicity of cells at the transplant site or to reduce proliferation of cells that have become tumorigenic at the transplant site), the method comprising: a) genetically modifying one or more (e.g., one, two, three, or more) cell division loci (CDLs) in a cell, where the CDLs are one or more loci whose transcription products are expressed by dividing cells (e.g., all dividing cells that contain one or more immunosuppressive transgenes), the genetic modification of the CDLs comprising one or more of: i) an ablation link (ALINK) system comprising a DNA sequence encoding a negative selection marker transcriptionally linked to a DNA sequence encoding the CDL; and i) an inducible exogenous activator of regulation (EARC) system comprising an inducible activator-based gene expression system operably linked to the CDL; and b) genetically modifying the cell to comprise at least one mechanism for providing local immunosuppression at the transplant site. c) transplanting the cell or cell population into a transplantation site in an allogeneic host; and d) maintaining the genetically modified cells comprising an ALINK system in the absence of an inducer of the negative selection marker to allow proliferation of the genetically modified cells comprising an ALINK system or exposing the cells comprising an ALINK system to an inducer of the negative selection marker to ablate and / or inhibit proliferation of the genetically modified cells comprising an ALINK system and / or exposing the genetically modified cells comprising an EARC system to an inducer of an inducible activator-based gene expression system to allow proliferation of the genetically modified cells comprising an EARC system or maintaining the cells comprising an EARC system in the absence of an inducer of an inducible activator-based gene expression system to prevent or inhibit proliferation of the genetically modified cells comprising an EARC system.

[0040] In an embodiment of this method, genetic modification of the CDL comprises performing targeted replacement of the CDL using one or more of: a) a DNA vector comprising an ALINK system; b) a DNA vector comprising an EARC system; and c) a DNA vector comprising an ALINK system and an EARC system, wherein the ALINK and / or EARC system are each operably linked to the CDL.

[0041] 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 generated through EARC-modified alleles.

[0042] 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 involved in one or more of the 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 CDLs are two or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5, and Eef2 / EEF2, preferably the CDLs are Cdk1 and Top2A or Cdk1 and Eef2.

[0043] 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.

[0044] In various embodiments of the method, the EARC system is a doxycycline-induced "dox-crosslinking" system, a coumate switch-induced system, an ecdysone-induced system, a radio-wave-induced system, or a ligand-reversible dimerization system; preferably, the EARC system is a dox-crosslinking system.

[0045] In an embodiment of this method, the genetically modified cell comprises a set of transgenes, each transgene encoding a cytoplasmic, membrane-bound, or locally acting gene product having one or more of the following functions: a) attenuating antigen-presenting cell activation and function, b) attenuating graft-attacking leukocyte activity or cytolytic function, c) attenuating macrophage cytolytic function and phagocytosis of allograft cells, d) inducing apoptosis in graft-attacking leukocytes, e) attenuating local inflammatory proteins, and f) protecting against leukocyte-mediated apoptosis.

[0046] In an embodiment of this 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 genes encoding a biological agent that acts as an agonist of PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8, or Serpin B9(Spi6).

[0047] In an embodiment of this 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 Serpin B9 (Spi6).

[0048] In an embodiment of this method, the set of transgene genes includes genes encoding PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8, and Serpin B9(Spi6), or a biological substance that acts as an agonist of PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8, and Serpin B9(Spi6).

[0049] In embodiments of this 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 genes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or genes encoding a biological substance that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.

[0050] In embodiments of the method, the TGF-β or biological agent acts locally within the graft environment. In embodiments, the TGF-β or biological agent acts locally within the graft environment with minimal systemic effects.

[0051] In various embodiments of the methods, the cells are stem cells, cells amenable to genome editing and / or a source of a therapeutic cell type (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 cells are selected from the group consisting of embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germline stem cells, pulmonary stem cells or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte progenitor cells, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, osteocytes, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, nasal cells, and the like. The cell types include neural killer cells, skeletal muscle cells, cardiac myocytes, erythroid-megakaryocyte cells, eosinophils, macrophages, T cells, islet 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 adipocytes, hormone-secreting cells, epidermal keratinocytes, epithelial cells, kidney cells, germ cells, skeletal joint synoviocytes, periosteal cells, perichondrocytes, 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, serous cells, somatic cells, or cells derived from the skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.

[0052] In various embodiments of the method, the cells are provided (e.g., injected) at or near the transplant site. In various embodiments of the method, the cells are provided (e.g., injected or transplanted) into a graft (e.g., injected or transplanted into a tissue or organ graft before, during, or after transplant). In some embodiments, the cells in which the transgene is expressed are cells of the graft (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)).

[0053] In various embodiments of the method, the allogeneic host is a mammal. In various embodiments of the method, the allogeneic host is a mouse or a human.

[0054] In various embodiments of the method, the host has a degenerative disease or condition that can be treated using cell therapy. In various embodiments, the disease or condition 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, neurological disease such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS, enzyme or hormone deficiency, metabolic disorder (e.g., lysosomal storage disorder, galactosemia, maple syrup urine disease, phenylketonuria, glycogen storage disease, mitochondrial disorder, Friedrich's ataxia, peroxisomal disorder, metal metabolism disorder, or organic acidemia. ), autoimmune diseases (e.g., psoriasis, systemic lupus erythematosus, Grave's 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 dystrophies, infectious diseases, hemophilia, degenerative diseases (e.g., Charcot-Marie-Tooth 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 (Fribrodisplasia ossificans progressiva) Ossificans Progressiava), infantile neuroaxonal dystrophy, keratoconus, keratoglobus, muscular dystrophy, neuronal ceroid lipofuscinosis, previous disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age-related diseases (e.g., atherosclerosis, cardiovascular disease), cardiovascular disease (e.g., angina pectoris, myocardial infarction), cataracts, osteoporosis or hypertension).

[0055] In some embodiments of any of the aforementioned 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 that is equal to or greater than the expression level of the corresponding endogenous gene in activated leukocytes (e.g., the expression level of the T cell, e.g., cloaking 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, or more times greater 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.

[0056] In some embodiments of any of the aforementioned 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 levels that exceed the expression level of the corresponding endogenous gene in wild-type stem cells (e.g., the expression level of the cloaking transgene is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1,000 or more times greater in cloaked cells compared to 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 that are higher (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 times higher) than the expression levels of the endogenous genes in wild-type stem cells (e.g., embryonic stem cells from the same species as the cloak 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 levels that are in the top 5% of gene expression for 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 levels that are in the top 1% of gene expression for 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 that are in the top 5% of gene expression for all genes in the ES cell genome.

[0057] In some embodiments of any of the aforementioned aspects, the PD-L1 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12.

[0058] In some embodiments of any of the foregoing aspects, the HLA-G(H2-M3) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 15.

[0059] In some embodiments of any of the foregoing aspects, the Cd47 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0060] In some embodiments of any of the foregoing aspects, the CD200 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0061] In some embodiments of any of the foregoing aspects, the FASLG (FasL) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:9.

[0062] In some embodiments of any of the foregoing aspects, the Ccl21 (Ccl21b) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1.

[0063] In some embodiments of any of the foregoing aspects, the Mfge8 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14.

[0064] In some embodiments of any of the foregoing aspects, the Serpin B9 (Spi6) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:7.

[0065] In some embodiments of any of the foregoing aspects, the IFNγR1 d39 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity) to the amino acid sequence of SEQ ID NO: 17.

[0066] In some embodiments of any of the foregoing aspects, the one or more transgenes are operably linked to a constitutive promoter, hi some embodiments, 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

[0067] In some embodiments of any of the foregoing aspects, the cell further comprises a transgene encoding a therapeutic agent (e.g., the cell is modified to further 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 a drug listed in Table 2. In some embodiments, the therapeutic agent is the wild-type version of a gene that is mutated in the subject (e.g., the wild-type version of a mutated gene associated with a disease or condition in the subject, e.g., a genetic mutation associated with cancer, an enzyme or hormone deficiency, a metabolic disorder, or a 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 radiogenic system, a cumate switch-inducible system, an ecdysone-inducible system, a destabilization 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 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

[0068] In another aspect, a population of genetically modified cells according to any of the above cells is provided.

[0069] In one aspect, a method is provided for providing local immunosuppression at a transplant site in an allogeneic host, the method comprising transplanting a genetically modified cell as described above or a population of genetically modified cells as described above into the transplant site in the allogeneic host.

[0070] In another aspect, the invention features a composition including the cells of the invention. In some embodiments, the composition further includes a pharmaceutically acceptable excipient.

[0071] Another aspect features a kit that includes a cell of the invention or a pharmaceutical composition of the invention.

[0072] Another aspect features a method of treating a disease or condition in a subject in need thereof by administering to the subject a cell of the invention or a composition of the invention. In some embodiments, the disease or condition 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 disorder, neurological disease such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS, enzyme or hormone deficiency, metabolic disorder (e.g., lysosomal storage disorder, galactosemia, maple syrup urine disease, phenylketonuria, glycogen storage disorder, mitochondrial disorder, Friedrich's ataxia, peroxisomal disorder, metal metabolism disorder, or organic acidemia), autoimmune disease (e.g., psoriasis, systemic lupus erythematosus, Grave's 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 disease, blood The subject is further characterized in that the subject is a neuropathic or inflammatory disease, such as rheumatoid arthritis, a neuropathic or inflammatory disease, a degenerative disease (e.g., Charcot-Marie-Tooth 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, keratoglobuclear, muscular dystrophy, neuronal ceroid lipofuscinosis, previous disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or an age-related disease (e.g., atherosclerosis, cardiovascular disease (e.g., angina pectoris, myocardial infarction), cataract, osteoporosis, or hypertension), or a disease or condition listed in Table 2, and / or a wild-type version of a gene that is mutated in the subject (e.g., a wild-type version of a mutated gene associated with a disease or condition in the subject, e.g., a gene 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 retinal dystrophy, and the therapeutic agent is a VEGF inhibitor (e.g., a soluble form of a VEG 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). parental , VEGF-Trap ΔB1 , VEGF-Trap ΔB2 , VEGF-Trap R1R2 , e.g., afliberept). In some embodiments, the disease or condition is osteoarthritis or rheumatoid arthritis, and the therapeutic agent is an anti-inflammatory biological agent (e.g., a TNFα inhibitor (e.g., adalimumab, etanercept, infliximab, golimumab, or sartolizumab), 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 condition is diabetes (e.g., type 1 diabetes or type 2 diabetes), and the therapeutic agent is insulin. In some embodiments, the disease or condition is hemophilia, and the therapeutic agent is factor VIII. In some embodiments, the disease or condition is a metabolic defect, and the therapeutic agent is a transgene having the nucleic acid sequence of a wild-type version of a gene that is mutated in the subject, or a transgene encoding an enzyme that is deficient in the subject.

[0073] In some embodiments of any of the foregoing aspects, the cells are differentiated into a lineage-restricted cell type prior to administration to a subject. In some embodiments, the disease or condition is myocardial infarction, and the cells are differentiated into cardiomyocytes. In some embodiments, the disease or condition is blindness, and the cells are differentiated into photoreceptor cells. In some embodiments, the disease or condition is spinal cord injury, Parkinson's disease, Huntington's disease, or Alzheimer's disease, and the cells are dissociated into neurons. In some embodiments, the disease or condition is multiple sclerosis, and the cells are differentiated into glial cells.

[0074] In some embodiments of any of the foregoing aspects, the cells are administered locally (e.g., injected or implanted) to a tissue or body site in need of the cells or therapeutic agent.

[0075] In some embodiments of any of the foregoing aspects, the cells are administered intravenously, subcutaneously, intramuscularly, transcutaneously, intradermally, parenterally, intraarterially, intravascularly, or by perfusion.

[0076] In some embodiments of any of the aforementioned aspects, the cells are administered by subcutaneous injection to produce cloaked subcutaneous tissue.

[0077] In some embodiments of any of the aforementioned aspects, the cells are administered as a tissue, hi some embodiments, the tissue is administered with a gel, a biocompatible matrix, or a cell scaffold.

[0078] In some embodiments of any of the aforementioned aspects, the cells are cultured at 25,000 to 5,000,000,000 cells (e.g., 2.5 x 10 4 , 5×10 4 , 7.5×10 4 , 1×10 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 6×10 5 , 6×10 5 , 7×10 5 , 8×10 5, 9×10 5 , 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 or 5 x 10 9 The amount of the antibody administered is 1000 cells / ml.

[0079] In some embodiments of any of the aforementioned aspects, the cells are between 800,000,000 and 100,000,000,000 cells (e.g., 8 x 10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 or 3 x 10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10, 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 or 1 x 10 11 The amount of the antibody administered is 1000 cells / ml.

[0080] In some embodiments of any of the aforementioned methods, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered before administration of the cells. In some embodiments, the additional therapeutic agent is administered after administration of the cells. In some embodiments, the additional therapeutic agent is administered simultaneously with administration of the cells. In some embodiments, the additional therapeutic agent is an immunosuppressant, a disease-modifying antirheumatic drug (DMARD), a biological 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, or kinelet. , certolizumab, etanercept, golimumab, infliximab, rituximab or tocilizumab, 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab, aminosalicylates, antibiotics, antihistamines, anti-TNFα, azathioprine, belimumab, beta interferon, calcineurin inhibitors, certolizumab, corticosteroids, cromolyn, cyclosporine A, cyclosporine, dimethyl fumarate, etanercept , fingolimod, fumarate, glatiramer acetate, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonists, long-acting beta-2 agonists, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, probiotics, retinoids, salicylic acid, short-acting beta-2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, toxin lizumab, ustekinumab or vedolizumab, bevacuzimab, ranibizumab or aflibercept), photodynamic therapy, photocoagulation, carbidopa-levodopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase inhibitors, anticholinergics, amantadine, deep brain stimulators, anticoagulants, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, angiotensin receptor neprilysin inhibitors, beta-blockers,Combination alpha and beta blockers, calcium channel blockers, cholesterol-lowering drugs, nicotinic acid, cholesterol absorption inhibitors, digitalis preparations, diuretics, vasodilators, dual antiplatelet therapy, cardiac procedures, antiviral compounds, nucleoside analog reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, antibacterial compounds, antifungal compounds, antiparasitic compounds, insulin, sulfonylureas, biguanides, meglitinides, thiazolidinediones , DPP-4 inhibitors, SGLT2 inhibitors, α-glucosidase inhibitors, bile acid sequestrants, aspirin, dietary therapy, clotting factors, desmopressin, clot-preserving drugs, fibrin sealants, physical therapy, coenzymes, bone marrow transplants, organ transplants, hemodialysis, hemofiltration, exchange transfusions, peritoneal dialysis, medium-chain triacylglycerols, miglustat, enzyme replacement therapy, checkpoint inhibitors, chemotherapy drugs, biological agents, radiation therapy, cryotherapy, hyperthermia, surgical resection or tumor tissue, or anti-cancer vaccines.

[0081] In some embodiments of any of the foregoing methods, the method further comprises controlling proliferation of the cells. In some embodiments, the cells comprise an ALINK system, and the method of controlling proliferation comprises i) maintaining the cells comprising the ALINK system in the absence of an inducer of the negative selection marker, thereby allowing proliferation of the cells comprising the ALINK system, or ii) exposing the cells comprising the ALINK system to an inducer of the negative selection marker, thereby ablating or inhibiting proliferation of the cells comprising the ALINK system. In some embodiments, the cells comprise an EARC system, and the method of controlling cell proliferation comprises i) exposing the cells comprising the EARC system to an inducer of an inducible activator-based gene expression system, thereby allowing proliferation of the cells comprising the EARC system, or ii) maintaining the cells comprising the EARC system in the absence of an inducer of the inducible activator-based gene expression system, thereby preventing or inhibiting proliferation of the cells comprising the EARC system.

[0082] In some embodiments of any of the foregoing methods, the cells are removed after completion of treatment. Cell removal can be by surgery (e.g., to remove transplanted tissue or organs or to remove cloaked subcutaneous tissue) or by use of ALINK and / or EARC systems. In some embodiments, one or more (e.g., one, two, three, four, or more) ALINK and / or EARC systems are used to eliminate all of the cloaked cells.

[0083] In another aspect, the present invention provides a cell of the present invention or a composition of the present invention for use in treating a disease or condition in a subject in need thereof. In some embodiments, the disease or condition 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, neurological disease such as Parkinson's disease, Alzheimer's disease, Huntington's disease and ALS, enzyme or hormone deficiency, metabolic disorder (e.g., lysosomal storage disorder, 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, Grave's 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, infection, hemophilia, degenerative diseases (e.g., Charcot-Marie-Tooth 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, keratoglobuloid, muscular dystrophy, neuronal ceroid lipofuscinosis, previous disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age-related diseases (e.g., atherosclerosis), cardiovascular diseases (e.g., angina pectoris, myocardial infarction), cataracts, osteoporosis or hypertension), or a disease or condition listed in Table 2.

[0084] In another aspect, the invention provides a cell of the invention or a composition of the invention for use in providing local immunosuppression at the site of transplantation in an allogeneic host.

[0085] In some embodiments of any of the foregoing aspects, the cells are transgenic for two of the following sets of 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 Serpin B9 (Spi6)). B9 (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 Serpin B9 (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); C200 and Mfge8; Cd200 and Serpin B9 (Spi6); FASLG (FasL) and Ccl21 (Ccl21b); FASLG (FasL) and Mfge8; FASLG (FasL) and Serpin B9 (Spi6); Ccl21 (Ccl21b) and Mfge8; Ccl21 (Ccl21b) and Serpin B9 (Spi6); or Mfge8 and Serpin B9 (Spi6)).

[0086]

[0013] In some embodiments of any of the foregoing aspects, the cells express three of the set 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)). 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 Serpin B9(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 Serpin B9 (Spi6); Cd47, Cd200, and FASLG (FasL); Cd47, Cd200, and Ccl21 (Ccl21b); Cd47, Cd200, and Mfge8; Cd47, C200, 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);

[0087] In some embodiments of any of the foregoing aspects, the cells express four of the set 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)). 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 Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Ccl21(Ccl21b), and Mfge8; PD-L1, HLA-G(H2-M3), Ccl21(Ccl21b), and Serpin B9(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); HLA-G(H2-M3), Ccl21(Ccl21b), Mfge8, and Serpin 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 Serpin B9 (Spi6); Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6); or FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6);

[0088] In some embodiments of any of the foregoing aspects, the cells express five of the set 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)). B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL) and Ccl21 (Ccl21b); PD-L1, HLA-G (H 2-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(H 2-M3), Cd200, FASLG(FasL) and Mfge8, PD-L1, HLA-G(H2-M3), Cd200, FASLG(FasL) and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd200, Ccl21(Ccl21b) and Mfge8; PD-L1, HLA-G(H2-M3), Cd200, Ccl21(Ccl21b) and Serpin B9(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 Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), FASLG (FasL), Mfge8 and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Ccl21(Ccl21b), Mfge8 and Serpin B9(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 cells express six of the set 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)). 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, 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));

[0089] In some embodiments of any of the foregoing aspects, the cells express seven of the set 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); PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Mfge8, 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, Cd47, 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)).

[0090] In some embodiments of any of the aforementioned aspects, the cells comprise all eight of the following set of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0091] 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 set of transgenes: HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0092] 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 set of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0093] 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 set of transgenes: HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0094] In some embodiments of any of the aforementioned aspects, the cells have not been modified to express PD-L1.

[0095] In some embodiments of any of the aforementioned aspects, the cells have not been modified to express FasL.

[0096] In some embodiments of any of the preceding aspects, the cells have not been modified to express TGF-β. In some embodiments of any of the preceding aspects, the cells have not been modified to express CTLA4 or CLTA4-Ig. In some embodiments of any of the preceding aspects, the cells have not been modified to express IDO. In some embodiments of any of the preceding aspects, the cells have not been modified to express IL-35. In some embodiments of any of the preceding aspects, the cells have not been modified to express IL-10. In some embodiments of any of the preceding aspects, the cells have not been modified to express VEGF. In some embodiments of any of the preceding aspects, the cells have not been modified to express NFκb decoy receptor F. In some embodiments of any of the preceding aspects, the cells have not been modified to express a soluble TNFR. In some embodiments of any of the preceding aspects, the cells have not been modified to express CCR7. In some embodiments of any of the preceding aspects, the cells have not been modified to express SOCS1. In some embodiments of any of the preceding aspects, the cells have not been modified to express HLA-E. In some embodiments of any of the aforementioned aspects, the cells have not been modified to express an siRNA directed against IL-12.

[0097] definition 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.

[0098] As used herein, the term "about" refers to a value that is no more than 10% above or below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0099] 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 a perceived insult. When leukocytes are activated, they proliferate, secrete cytokines, differentiate, present antigens, become more polarized, more phagocytic, and / or more cytotoxic. Factors that stimulate immune cell activation include proinflammatory cytokines, pathogens, and non-self antigen presentation. Activated leukocytes can be isolated from lymphoid organs. Leukocytes, such as T cells, can also be activated in vitro using anti-CD3 / CD28 beads or other methods used 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)).

[0100] As used herein, "allogeneic" means cells, tissues, DNA, or factors taken from or derived from a different subject of the same species.

[0101] As used herein, the term "stem cell" refers to a cell that can differentiate into one or more specialized cells and has the capacity for self-renewal. Stem cells include pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), as well as multipotent stem cells, such as umbilical cord blood stem cells, mesenchymal stromal cells, and adult stem cells, found in various tissues. The term "stem cell" also includes cells amenable to genome editing, cells that can serve as a source of therapeutic cell types (e.g., lineage-restricted or terminally differentiated cells used in cell therapy or cells of a desired target tissue), and "artificial" cells derived from stem cells with stem cell properties (e.g., pluripotency or multipotency or self-renewal).

[0102] As used herein, the terms "embryonic stem cells" and "ES cells" refer to embryo-derived totipotent or pluripotent stem cells derived from the inner cell mass of a blastocyst that can be maintained in in vitro culture under appropriate conditions. ES cells can differentiate 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 proliferate indefinitely under appropriate in vitro culture conditions. See, e.g., Thomson et al., Science 282:1145 (1998).

[0103] As used herein, the terms "induced pluripotent stem cells," "iPS cells," and "iPSCs" refer to pluripotent stem cells that can be derived directly from differentiated somatic cells. For example, human iPS cells can be generated by introducing a specific set of reprogramming factors into non-pluripotent cells, including Oct3 / 4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, l-Myc, n-Myc), Krüppel-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 generated by the use of lineage-determining factors, such as miRNAs, which are small molecules that mimic the action of transcription factors. Human iPS cells are characterized by their ability to differentiate into any of the three vertebrate germ layers, e.g., endoderm, ectoderm, or mesoderm. Human iPS cells are also characterized by their ability to proliferate indefinitely under appropriate in vitro culture conditions (see, e.g., Takahashi and Yamanaka, Cell 126:663 (2006)).

[0104] As used herein, the term "attenuating antigen-presenting cell activation and function" refers to a transgene encoding a gene product whose function is to inhibit the activation of antigen-presenting cells or the ability of antigen-presenting cells to promote the activation of leukocyte-attacking grafts (Fiorentino et al., J Immunol. 146:3444-51 (1991); Salio et al., Eur J Immunol. 29:3245-53 (1999)). In embodiments, attenuated antigen presenting cell activation and function refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% decrease in APC activation and function relative to a control (e.g., as determined using an assay for antigen presenting cell activation, such as reduced proliferation, reduced secretion of pro-inflammatory 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, e.g., TNFα), interferon gamma (IFNγ), and granulocyte-macrophage colony-stimulating factor (GMCSF) (which can be measured using ELISA or Western blot analysis of a patient sample such as culture medium or blood)), or cell surface markers (e.g., For example, decreased levels of 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 for the 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 attenuation of antigen-presenting cell activation and function are known in the art. Examples of gene products that attenuate 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 "cloaking" or "cloaked" genes.

[0105] As used herein, the term "mitigating the activity or cytolytic function of graft-attacking leukocytes" refers to a transgene encoding a gene product whose function is to inhibit or prevent the activity or cytolytic function of graft-attacking leukocytes in the vicinity of allograft 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, attenuating activation or cytolytic function of graft-attacking leukocytes refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% decrease in leukocyte activation or cytolytic function relative to a control (e.g., as measured using an assay for leukocyte activation, e.g., decreased proliferation, decreased pro-inflammatory cytokines (e.g., interleukin-1 (IL-1, e.g., IL-1β), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL- secretion of IL-13, IL-18, tumor necrosis factors (TNF, e.g., TNFα), interferon gamma (IFNγ), and granulocyte-macrophage colony-stimulating factor (GMCSF), which can be measured using ELISA or Western blot analysis of patient samples such as culture medium or blood) or decreased polarization (e.g., levels of IL-12, TNF, IL-1β, IL-6, IL-23, MARCO, MHC-II, CD86, iNOS, CXCL9, and CXCL10 in macrophages or monocytes). Decreased levels of Th1-specific markers (e.g., T-bet, IL-12R, STAT4), chemokine receptors (e.g., CCR5, CXCR6, or CXCR3); or decreased levels of Th2-specific markers (e.g., CCR3, CXCR4, STAT6, GATA3, or IL-4Rα) in T cells, which can be assessed using flow cytometry, immunohistochemistry, in situ hybridization, qPCR, or Western blot analysis of cell surface markers or intracellular proteins and ELISA or Western blot analysis for secreted proteins), or using assays for cytolytic function (e.g., by incubating leukocytes with target cell lines pre-coated with antibodies against surface markers expressed by the target cell line and measuring the number of viable target cells with fluorescent viability staining, or by measuring secretion of cytolytic granules (e.g., perforin, granzymes, or other cytolytic proteins released by immune cells) from leukocytes). Methods for determining the activation or attenuation of cytolytic function of graft-attacking leukocytes are known in the art.Examples of gene products that mitigate the activation or cytolytic function of graft-attacking leukocytes include, but are not limited to, PD-L1, HLA-G(H2-M3), Cd39, Cd73, and Lag3. Such transgenes may be referred to herein as "cloaking" or "cloaked" genes.

[0106] As used herein, the term "mitigating macrophage cytolytic function and phagocytosis of allograft cells" refers to a transgene encoding a gene product whose function is to inhibit or prevent macrophage cytolytic function and / or phagocytosis of allograft 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 embodiments, attenuated macrophage cytolytic function and phagocytosis of allograft cells refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% decrease in macrophage cytolytic function and / or phagocytosis of allograft cells relative to a control (e.g., as determined using an assay for macrophage cytolytic function (e.g., by incubating macrophages with a target cell line pre-coated with an antibody against 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 number of viable target cells with fluorescent viability staining, ... This can be determined by measuring secretion of cloaking proteins or using assays for macrophage phagocytosis (e.g., culturing macrophages with target cell lines pre-coated with fluorescent beads or antibodies against surface antigens expressed by the target cell line and measuring the internal fluorescence of the immune cells or quantifying the number of beads or engulfed cells). Methods for determining attenuation of macrophage cytolytic function and phagocytosis of allograft cells are known in the art. Examples of gene products that attenuate macrophage cytolytic function include, but are not limited to, Cd47, Cd200, Mfge8, and Il1r2. Such transgenes may be referred to herein as "cloaking" or "cloaked" genes.

[0107] As used herein, the term "inducing apoptosis in graft-attacking leukocytes" refers to a transgene encoding a gene product whose function is to kill graft-attacking leukocytes in the vicinity of graft cells (Huang et al., Proc Natl Acad Sci US A. 96:14871-6 (1999); Suzuki et al., Proc Natl Acad Sci US A. 97:1707-12 (2000); Simon et al., Proc Natl Acad Sci US A. 98:5158-63 (2001)). In embodiments, induction of apoptosis in graft-attacking leukocytes refers to an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% in the induction of apoptosis in graft-attacking leukocytes relative to a control (e.g., as determined using an assay for apoptosis, such as the use of TUNEL staining, caspase staining, or Annexin V staining, or a fluorescent viability stain). Methods for determining the induction of apoptosis in graft-attacking leukocytes are known in the art. Examples of gene products capable of inducing apoptosis in graft-attacking leukocytes include, but are not limited to, FASLG (FasL) and Tnfsf10. Such transgenes may be referred to herein as "cloaking" or "cloaked" genes.

[0108] As used herein, the term "mitigating local immune proteins" refers to a transgene that encodes a gene product whose function is to inhibit the activity of a local protein whose function is to promote graft-attacking leukocyte accumulation and / or their cytolytic function (Felix et al., Nat Rev Immunol. 17:112-29 (2017)). In embodiments, amelioration of local immune proteins refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% decrease in local inflammatory proteins relative to a control (e.g., as determined using an assay for inflammatory proteins that promote leukocyte activation or migration to sites 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 amelioration of local inflammatory proteins are known in the art. Examples of gene products that attenuate local inflammatory proteins include, but are not limited to, PD-L1, Il1r2, and Ackr2. Such transgenes may be referred to herein as "cloaking" or "cloaked" genes.

[0109] As used herein, the term "protects against leukocyte-mediated apoptosis" refers to a transgene that encodes a gene product whose function is to inhibit any cellular components that may induce apoptosis or cell lysis of allograft cells (Abdullah et al., J Immunol. 178:3390-9 (2007)). In embodiments, protection against leukocyte-mediated apoptosis refers to at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% reduction in leukocyte-mediated apoptosis 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 an antibody against 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 number of cytolytic granules (e.g., perforin, granzymes, or other cytolytic proteins released from immune cells) released from the leukocytes). Methods for determining protection against 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 "cloaking" or "cloaked" genes.

[0110] As used herein, the term "biological material" refers to an engineered polypeptide and corresponding encoding DNA that can be expressed as a transgene. The polypeptide can agonize 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 embodiments, agonist function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the function relative to the function of a control. In embodiments, antagonist function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the function relative to the function of a control.

[0111] As used herein, the term "operably linked" refers to a first molecule linked to a second molecule, the molecules arranged such that the first molecule affects the function or expression of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule of interest if the promoter controls the transcription of the transcribable polynucleotide molecule in a cell. Furthermore, two portions of a transcriptional regulatory element are operably linked to each other if they are linked such that the transcriptional activation function of one portion is not adversely affected by the presence of the other portion. Two transcriptional regulatory elements can be operably linked to each other by a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or can be operably linked to each other without the presence of any intervening nucleotides.

[0112] As used herein, the term "promoter" refers to a recognition site on DNA that is bound by an RNA polymerase, which drives transcription of the allograft.

[0113] "Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be accomplished by a variety of methods within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters, including any algorithm for aligning sequences, necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of example, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or relative to a given nucleic acid or amino acid sequence B (which can alternatively be expressed as a given nucleic acid or amino acid sequence A having a particular percent sequence identity to, with, or relative to a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(ratio X / Y) where 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 Y is the total number of nucleic acids in B. It is understood that if nucleic acid or amino acid sequence A is not equal in length to nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0114] As used herein, the term "pharmaceutical composition" refers to a mixture comprising a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and / or carriers, that is administered to a subject, e.g., a mammal, e.g., a human, to prevent, treat or control a particular disease or condition that the subject has or may develop.

[0115] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of a subject, e.g., a mammal (e.g., a human), without excessive toxicity, irritation, allergic response and / or other problem complications, commensurate with an ideal benefit / risk ratio.

[0116] As used herein, the term "wild type" refers to the genotype that has the highest frequency for a particular gene in a given organism.

[0117] As used herein, the terms "cell division locus," "cell division loci," and "CDL" refer to a genomic locus whose transcription product is expressed by dividing cells. When a CDL comprises a single locus, the absence of CDL expression in a cell (or its derivative) means that the cell is either ablated in the absence of CDL expression, or that cell proliferation is prevented or impaired in the absence of CDL expression, thereby preventing tumor initiation and / or formation. When a CDL comprises multiple loci, the absence of expression by all or a subset of the loci in a cell (or its derivative) means that the cell is either ablated in the absence of CDL expression, or that cell proliferation is prevented or impaired in the absence of CDL expression, thereby preventing tumor initiation and / or formation. A CDL may or may not be expressed in non-dividing and / or non-proliferating cells. A CDL may be endogenous to a host cell or may be a transgene. When the CDL is a transgene, it can be derived from the same or a different species as the host cell, or it can be of synthetic origin. In one embodiment, the CDL is a single locus that is transcribed during cell division. For example, in one embodiment, the single locus CDL is CDK1. In one embodiment, the 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, which may have overlapping functions (Fernandez-Miranda et al., 2011). Cell division and cell proliferation are terms that may be used interchangeably herein.

[0118] As used herein, the terms "normal rate of cell division," "normal rate of cell division," "normal rate of cell proliferation," and "normal rate of cell proliferation" refer to the rate of cell division and / or proliferation typical of non-cancerous, healthy cells. Normal rates of cell division and / or proliferation may be cell type specific. For example, it is widely accepted that cell numbers in the epidermis, intestine, lung, blood, bone marrow, thymus, testis, uterus, and mammary gland are maintained by high rates of cell division and cell death. In contrast, cell numbers in the pancreas, kidney, cornea, prostate, bone, heart, and brain are maintained by low rates of cell division and cell death (Pellettieri and Sanchez Alvarado, 2007).

[0119] As used herein, the terms "inducible negative growth effector" and "iNEP" refer to genetic modifications that facilitate the use of CDL expression to control cell division and / or proliferation by: i) inducibly silencing or preventing CDL expression, thereby preventing cell division and proliferation; ii) inducibly ablating CDL-expressing cells (i.e., killing at least a portion of the proliferating cells); or iii) slowing the rate of cell division relative to the normal cell division rate of the cells, so that the rate of cell division is not rapid enough to contribute to tumor formation.

[0120] As used herein, the terms "ablation link" and "ALINK" refer to an example of an iNEP that includes a transcriptional link between a CDL and a sequence encoding a negative selection marker. The ALINK modification allows a user to inducibly kill proliferating host cells that include the ALINK by exposing the ALINK-modified cells to an inducer of the negative selection marker, or to inhibit host cell proliferation by killing at least a portion of the proliferating cells. For example, cells modified to include an ALINK in the CDL can be treated with an inducer of the negative selection marker (e.g., a prodrug) to ablate proliferating cells or inhibit cell proliferation by killing at least a portion of the proliferating cells.

[0121] As used herein, the terms "exogenous activator of CDL regulation" and "EARC" refer to an example of an iNEP that includes a mechanism or system that facilitates exogenous modification of non-coding or coding DNA transcription or corresponding translation via an activator. EARC modification allows users to stop or inhibit the division of cells containing EARC by removing an inducer that enables transcription and / or translation of EARC-modified CDLs from EARC-modified cells. For example, an inducible activator-based gene expression system can be operably linked to a CDL and used to exogenously control CDL expression or CDL translation, thereby requiring the presence of a drug-inducible activator and a corresponding inducer for CDL transcription and / or translation. In the absence of the inducer, cell division and / or proliferation will be stopped or inhibited (e.g., slowed to a normal cell division rate). For example, CDL Cdk1 / CDK1 can be modified to contain a dox-bridge such that expression of CDL Cdk1 / CDK1 and cell division and proliferation are only possible in the presence of an inducer (e.g., doxycycline).

[0122] As used herein, the term "growth antagonist system" refers to a natural or engineered compound, the presence of which inhibits (fully or partially) cell proliferation.

[0123] As used herein, the term "dox-bridge" refers to a mechanism for separating promoter activity from a target transcription region by expressing rtTA from an endogenous or exogenous promoter (Gossen et al., 1995) and placing transcription of the target region under the control of a TRE. As used herein, "rtTA" refers to the reverse tetracycline transactivator of the tetracycline-inducible system (Gossen et al., 1995), and "TRE" refers to a promoter consisting of a TetO operator sequence upstream of a minimal promoter. When rtTA binds to the TRE promoter in the presence of doxycycline, transcription of the locus downstream of the TRE promoter increases. The rtTA sequence can be inserted in the same transcription unit as the CDL or at a different location in the genome, as long as the permissive or nonpermissive state of transcriptional expression of the target region is controlled by doxycycline. Dox-bridge is an example of an EARC.

[0124] As used herein, the term "fail-safe cell" refers to a cell that contains one or more homozygous, heterozygous, hemizygous, or compound heterozygous ALINK or EARC modifications in one or more CDLs (e.g., at least two, three, four, or five). A fail-safe cell can contain either ALINK or EARC modifications, or both ALINK and EARC modifications (e.g., ALINK and EARC modifications in different CDLs or a single CDL).

[0125] As used herein, the term "fail-safe" refers to the property of a cell that is unlikely to exhibit uncontrolled (e.g., tumorigenic) growth. A cell can be considered "fail-safe" if its growth is under the control of a negative regulator or inducer and it is unlikely to lose the activity of the growth-controlling system through genetic mutation. The fail-safe volume depends on the number of ALINKs and the number of CDLs that target the ALINKs (e.g., a cell with homozygous modifications of two different CDLs has a larger fail-safe volume than a cell with heterozygous modifications of a single CDL (e.g., it is less likely to lose all of the growth-controlling systems through genetic mutation)). Fail-safe properties are further described in Table 3.

[0126] The patent or patent application file contains at least one drawing with 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.

[0127] These and other features of the present disclosure will become more apparent in the following detailed description, in which reference is made to the accompanying drawings. [Brief explanation of the drawings]

[0128] [Figure 1A-D] Representative images showing the expression of cloaking proteins (Cd200 (Figure 1A), FasL (Figure 1B), H2-M3 (Figure 1C), and Cd47 (Figure 1D)) in the C57BL / 6 mouse embryonic stem cell line C2 using immunohistochemistry. [Figure 2A-E] Flow cytometry plots showing T cell activation using splenocytes (Figure 2A), wt B16 melanoma cells (Figure 2B), cloaked B16 melanoma cells (Figure 2C), wt ES cells (Figure 2D), and cloaked ES cells (Figure 2E) in a mixed lymphocyte reaction. [Figure 3A-B] Schematic diagram and images showing that cloaked (Figure 3B) B16F10 cancer cells in an allogeneic model are protected from rejection compared to their WT counterparts (Figure 3A). Representative images of non-cloaked cells in C57BL / 6 (n=5) and non-cloaked cells in FVB / N (n=4) (Figure 3A); cloaked cells in C57BL / 6 (n=5) and cloaked cells in FVB / N (n=6) (Figure 3B). [Figure 4] FIG. 1 is a schematic diagram showing that cloaked embryonic stem cells form tumors in syngeneic B6 mouse recipients (upper panel) and FVB allogeneic recipients (lower panel). [Figure 5A-C]5A and 5B are a series of photographs showing allogeneic mice bearing teratomas formed by subcutaneous injection of clonal C57BL / 6 ES cells. Red arrows indicate the teratomas. Figure 5A shows a teratoma in a C3H mouse, Figure 5B shows a teratoma in an FVB / N mouse, and Figure 5C shows a teratoma in a CD1 mouse. [Figure 6] 1 is a schematic series of images showing that animals with cloak tissue are immunocompromised. [Figure 7] 10 is a series of images of FVB / N mice showing further results indicating that animals with cloak cells are not immune compromised. [Figure 8A-C] Figure 8 shows transgene expression in clone failsafe containing embryonic stem cells derived from C57BL / 6 mice: Figure 8A shows FasL expression, Figure 8B shows Ccl21b expression, and Figure 8C shows Cd200 expression. [Figure 8D-F] Figure 8 shows transgene expression in clone failsafe containing embryonic stem cells derived from C57BL / 6 mice. Figure 8D shows Cd47 expression, Figure 8E shows Mfge8 expression, and Figure 8F shows Spi6 expression. [Figure 8G-H] Figure 8G shows transgene expression in clone fail-safe containing embryonic stem cells derived from C57BL / 6 mice. Figure 8G shows H2-M3 expression, and Figure 8H shows PD-L1 expression. [Figure 9] Figure 1 shows a series of graphs showing cloaking transgene expression in ES cell clones. Each cloaking transgene is shown in a different color. Concentric circles represent expression levels on a log10 scale. The thick black circle represents 1x expression normalized to a positive control (activated leukocytes isolated from mouse lymphoid organs), and the next outer rings represent 10x and 100x expression compared to the positive control, respectively. The innermost ring is 0.1x expression compared to the positive control. Clones NT2 and 15 (shown in red rectangles) had the highest expression of the cloaking gene. These clones survived in allogeneic hosts. [Figure 10]1 is a graph showing the expression of cloaking transgenes among the whole genome gene expression level distribution for the whole genome of ES cells. All eight cloaking transgenes in the NT2 cell line and NT2-derived teratomas have expression levels in the top 5% of all genes in the ES cell genome, and five of the cloaking transgenes have expression levels in the top 1% of all genes in the ES cell genome. The transgene expression levels in the NT2 cell line and NT2-derived teratomas successfully achieved allograft resistance. [Figure 11A-B] Figure 11B is a photograph showing a C57BL / 6-derived teratoma in an FVB / N mouse. The transgenic line, NT2, resulted in teratomas in 9 of 10 injection sites. Images were taken 3 months after injection. Figure 11B is a magnified view of the teratoma indicated by the arrow in Figure 11A. [Figure 12A-B] 12A and 12B are graphs showing teratoma tumor size in syngeneic (FIG. 12A) and allogeneic (FIG. 12B) mice treated with ganciclovir. [Figure 13A-B] FIG. 13 is a series of photomicrographs showing that cloaked embryonic stem cells injected into both syngeneic (FIG. 13A) and allogeneic (FIG. 13B) hosts can differentiate into all three cell lineages. [Figure 14A-D] 14A, 14B, and 14C are photomicrographs showing the formation of all three germ layers in teratomas formed by subcutaneous injection of cloaked ES cells into mice. Figures 14A, 14B, and 14C show the three germ layers (ec = ectoderm, shown in Figure 14A; en = endoderm, shown in Figure 14C; me = mesoderm, shown in Figure 14B). Figure 14D shows blood vessels indicated by red arrows, confirming that the tissue is well vascularized. [Figure 15] FIG. 1 is a schematic diagram showing the construction of vectors expressing target genes essential for allo-resistance. [Figures 16A-H]Fluorescence micrographs showing the expression of proteins encoded by cloaking transgenes in ES cells: Figure 16A shows the expression of PD-L1, Figure 16B shows the expression of CD200, Figure 16C shows the expression of CD47, Figure 16D shows the expression of FasL, Figure 16E shows the expression of H2-M3, Figure 16F shows the expression of Ccl21, Figure 16G shows the expression of Mfge8, and Figure 16H shows the expression of Spi6. [Figure 17A-B] 17A and 17B are micrographs showing that cloaked ES cells have typical ES cell morphology (FIG. 17A) and express the ES cell marker alkaline phosphatase (FIG. 17B). [Figure 18A-B] Fluorescence micrographs showing the expression of pluripotent ES cell markers (Oct4 (FIG. 18A) and SSEA1 (FIG. 18B)) in cloak ES cells. The insets in FIGS. 18A-18B show single-channel images of fluorescence micrographs for ES cell markers (Oct4 and SSEA) and DAPI, which labels nuclei, demonstrating that staining for the ES cell markers colocalizes with cloak cells. [Figure 19] Schematic diagram showing the immune process inhibited by the cloaking transgene (top) and the expression cassette used to express the cloaking transgene in ES cells (bottom). [Figure 20] 1 is a series of graphs showing the effect of interferon gamma (IFNγ) on MHC levels in ES cells, demonstrating that IFNγ increased MHC levels in wild-type ES cells and 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), and that IFNγR1 d39 completely inhibited IFNγ-mediated upregulation of MHC in ES cells. DETAILED DESCRIPTION OF THE INVENTION

[0129] Cell and Method Description For example, methods are featured for providing local immunosuppression at the transplant site using tools and cells such as genetically modified cells when the cells are transplanted into an allogeneic host. The genetically modified cells contain one or a set of transgenes, each transgene encoding a cytoplasmic, membrane-bound, or locally acting gene product whose function is to mitigate host immune system function (e.g., graft-attacking leukocyte and NK cell activation) or act as a defense mechanism against leukocyte attack.

[0130] A variety of cytoplasmic, membrane-bound or locally acting immune factors have been found to regulate local immune compartments and populations. 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 US A.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. 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 Sciences of the United States of America.98:11515-20(2001);Zhang et al. al.,Immunity.24:451-61(2006);Soriano et al.,Lung Immune factors such as IL-16, ...

[0131] The present inventors have modified allogeneic cells through the use of specific immunomodulatory factors introduced into cells or cell populations. The modified cells avoid immune rejection through simultaneous modulation of many different local immune pathways. Such genetically engineered cells can be transplanted "off the shelf" into many recipients, regardless of genetic background, without rejection by the recipient's immune system. This immunomodulatory approach overcomes the need for systemic immunosuppression of the transplant recipient, which can be dangerous to the recipient. Thus, although immunosuppressants can be administered to patients receiving the modified cells described herein, this treatment does not need to include the administration of immunosuppressants. This immunomodulatory approach also overcomes the costly and impractical methodologies of deriving patient-specific iPS cells, engineering regulatory cells, or inducing chimerism via hematopoietic cell transplantation (HCT).

[0132] The cells can be genetically modified to express a set of transgenes that encode cytoplasmic, membrane-bound, or locally acting gene products whose function is to mitigate host immune system function (e.g., graft-attacking leukocyte and NK cell activation) or act as a defense mechanism against an immune response (e.g., leukocyte attack). The set of transgenes can be selected from genes that play a role in the gene regulatory pathways described above. Such genes include, but are not limited to, those provided in Table 1.

[0133] [Table 1]

[0134] The CC motif chemokine ligand 21 (Ccl21) is expressed by local lymph nodes, where Ccl21 acts to attract activated antigen-presenting cells (APCs). This important function provides an opportunity to "reverse" APC migration by overexpressing this gene on transplanted cells. Indeed, some melanomas express Ccl21 and are able to recruit CCR7+ cells, which then reorganize parts of their tumor stroma as "self." This is due to the CD4 + It leads to stromal remodeling that supports the recruitment and maintenance of Tregs (Zindl et al., Science. 328:697-8 (2010)). Indeed, expression of Ccl21 on tumors can protect co-transplanted Ccl21-deficient tumor cells from rejection in syngeneic allograft settings (Shields et al., Science. 328:749-52 (2010)). Ccl21b is the murine ortholog of human Ccl21.

[0135] The amino acid sequences of mouse and human Ccl21 are as follows: Mouse Ccl21 [ka] Human Ccl21 [ka]

[0136] Expression of Cd47 in the umbilical cord can promote the development of hyporesponsive T cells (Avice et al., J Immunol. 167:2459-68 (2001)). Erythrocytes also upregulate Cd47 to avoid dendritic cell activation due to their lack of "self" presentation (van den Berg et al., Immunity. 43:622-4 (2015)). More recently, increased expression of human Cd47 has been shown to increase engraftment in pig-to-human hematopoietic cell transplants in a mouse model (Tena et al., Am J Transplant. 14:2713-22 (2014)).

[0137] The amino acid sequences of mouse and human Cd47 are as follows: Mouse Cd47 [ka] Human Cd47 [ka]

[0138] Cd200 is also an important immunoregulatory molecule, and its increased expression can reduce the severity of allograft rejection, autoimmune, and allergic diseases (Gorczynski et al., J Immunol. 172:7744-9 (2004)). In vitro, APC expression of Cd200 has been shown to suppress the production of interferon-γ (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 of skin and cardiac allografts in mice by promoting Foxp3+ Treg cells (Gorczynski et al., Transplantation. 98:1271-8 (2014)).

[0139] The amino acid sequences of mouse and human Cd200 are as follows: Mouse Cd200 [ka] Human Cd200 [ka]

[0140] Spi6 is an endogenous inhibitor of granzyme B, a cytotoxic effector molecule released by activated CD8+ T cells (Sun et al., J Biol Chem. 272:15434-41 (1997)). Some data suggest that mesenchymal stem cells (MSCs) escape immune rejection by upregulating this molecule (El Haddad et al., Blood. 117:1176-83 (2011)). It has recently been demonstrated that the ability of dendritic cells to present antigens to cytotoxic T cells without being killed through contact-mediated cytotoxicity is mediated by Spi6 (Lovo et al., J Immunol. 188:1057-63 (2012)). Spi6 is also known as Serpin B9.

[0141] The amino acid sequences of mouse Spi6 and its human counterpart Serpin B9 are as follows: Mouse Spi6 [ka] Human Serpin B9 [ka]

[0142] Activated cytotoxic Cd8+ can kill target cells through the expression of FasL, which binds to the FAS receptor and activates caspase-mediated apoptosis in target cells. However, many tumors have developed a "counterattack" by upregulating FasL on their surface (Chen et al., J Immunol. 171:1183-91 (2003)). Selective expression of FasL in the vasculature of human and mouse solid tumors is associated with rare Cd8+ T cell infiltration and a predominance of FoxP3+ Treg cells (Motz et al. Nat Med. 20:607-15 (2014)). More recently, B lymphocytes have also been shown to use FasL expression to kill T helper cells during the effector phase of the immune response (Lundy et al., Front Immunol. 6:122 (2015)). FasL is the mouse orthologue of human FASLG.

[0143] The amino acid sequences of mouse FasL and the human counterpart FASLG are as follows: Mouse FasL [ka] Human FASLG [ka]

[0144] PD-L1 is an important immunoregulatory molecule that binds to programmed cell death (PD-1). PD-1 is expressed on T cells and binds to PD-L1, resulting in T cell anergy (MacDonald et al., J Immunol. 126:1671-5 (1981)).

[0145] The amino acid sequences of mouse and human PD-L1 are as follows: Mouse PD-L1 [ka] Human PDL1 (CD274) [ka]

[0146] An inflammatory environment, such as that induced by allografts, attracts macrophages and inflammatory monocytes, among many other innate immune cells. Milk fat globule epidermal growth factor 8 (Mfge-8) is expressed by many murine tumors (Neutzner et al., Cancer Res. 67:6777-85 (2007)) and has been shown to contribute to local immunosuppression by polarizing monocytes into suppressive M2-like macrophages (Soki et al., J Biol Chem. 289:24560-72 (2014)).

[0147] The amino acid sequences of mouse and human MFGE-8 are as follows: Mouse MFGE8 [ka] Human MFGE8 [ka]

[0148] The potent killing potential of NK cells is also crucial in graft rejection. NK cells can kill target cells lacking MHC class I molecules and other cells in inflammatory settings. H2-M3, the 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 through the binding of HLA-G, an immunosuppressive receptor on both NK and T cells (Carosella et al., Adv Immunol. 127:33-144 (2015)). H2-M3 is the mouse ortholog of human HLA-G.

[0149] The amino acid sequences of mouse H2-M3 and its human counterpart HLA-G are as follows: Mouse H2-M3 [ka] Human HLA-G [ka]

[0150] A transgene set 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 the cell. The cell can be, for example, a stem cell or a cell amenable to genome editing, such as a cell that can be used in therapy and / or differentiated 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 transgene set can include one, two, three, four, five, six, seven, or all eight of these genes, or at least one, at least two, at least three, at least four, at least five, at least six, or at least seven 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 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) using methods known to those skilled in the art. For example, one way to localize TGF-β to the membrane is to coexpress TGF-β with an additional transgene encoding the LRRC32 protein or any other polypeptide that results in localization of TGF-β 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)).

[0151] The amino acid sequence of IFNγR1 d39 is as follows: [ka]

[0152] The gene can be a human gene or a mouse gene. In embodiments, the gene is of the same species as the recipient of the allograft recipient into which the cells will be transplanted. In embodiments, the gene is of any species in which the function of the gene is conserved or in which the engineered biological material has an agonist function of the endogenous counterpart. Methods for introducing and expressing these transgenes in cells are described herein and are known to those of skill in the art. Cells expressing these transgenes may be referred to as "cloaked" due to their ability to avoid allogeneic rejection without systemic immunosuppression and without the need for immunosuppressive drugs.

[0153] It is contemplated herein that a population of cells derived from the above cloak cells may also be used to generate local immunosuppression when transplanted into the transplant site of an allogeneic recipient.

[0154] Before or after generating the cloak cells of the present disclosure, cells can be first modified to be fail-safe cells. Fail-safe cells use cell division loci (CDLs) to control cell proliferation in animal cells. A CDL, as provided herein, is a locus whose transcription product is expressed during cell division. A CDL can be genetically modified to include a gene expression system based on a negative selection marker and / or an inducible activator, as described herein, allowing users to enable, ablate, and / or inhibit proliferation of genetically modified cells by adding or removing the appropriate inducer. Methods for making and using fail-safe cells are described, for example, in WO 2016 / 141480, the entire teachings of which are incorporated herein by reference. Cells can be first made fail-safe and then cloaked. Alternatively, cells can be first cloaked and then made fail-safe.

[0155] The cells may be vertebrate cells, e.g., mammalian cells such as human or mouse cells. The cells may also be vertebrate stem cells, e.g., mammalian stem cells such as human or mouse stem cells. Preferably, the cells or stem cells are amenable to genetic modification. Preferably, the cells or stem cells are amenable to genetic modification. Preferably, the cells or stem cells are considered by the user to have therapeutic value, meaning that the cells or stem cells can be used to treat a disease, disorder, defect, or injury in a subject in need thereof.

[0156] In some embodiments, the cell is a stem or progenitor cell (e.g., an iPSC, embryonic stem cell, hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem cell or progenitor cell, germline stem cell, lung stem cell or progenitor cell, mammary stem cell, olfactory adult stem cell, hair follicle stem cell, multipluripotent stem cell, amniotic stem cell, umbilical cord blood stem cell, or neural stem or progenitor cell). In some embodiments, the stem cell is an adult stem cell (e.g., a somatic stem cell or a tissue-specific stem cell). In some embodiments, the stem or progenitor cell is capable of differentiation (e.g., the stem cell is totipotent, pluripotent, or multipotent). In some embodiments, the cell is isolated from embryonic or neonatal tissue. In some embodiments, the cells are fibroblasts, monocyte progenitor cells, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, progenitor cells, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, bone cells, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, or natural killer cells. In some embodiments, the cells are engineered (e.g., converted or differentiated) into myocytes, erythroid megakaryocytic cells, eosinophils, iPS cells, macrophages, T cells, islet beta cells, neurons, cardiomyocytes, blood cells, endocrine progenitor cells, exocrine progenitor cells, ductal cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, bile duct cells, or brown adipocytes. In some embodiments, the cell is a muscle cell (e.g., a skeletal, smooth, or cardiac muscle cell), an erythroid megakaryocytic cell, an eosinophil, an iPS cell, a macrophage, a T cell, an islet 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 cells are hormone-secreting cells (e.g., cells that secrete insulin, oxytocin, endorphins, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormones, bombesin, cholecystokinin, testosterone, estrogen or progesterone, renin, ghrelin, amylin, or pancreatic polypeptide), epidermal keratinocytes, epithelial cells (e.g., exocrine epithelial cells, thyroid epithelial cells, keratinizing epithelial cells, gallbladder epithelial cells, or cells secreting cells from the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, or 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 derived from a human or other mammal (e.g., a rodent, non-human primate, bovine, or porcine cell). It is contemplated herein that cloak cells may be useful in cell-based therapies where it may be desirable to avoid allogeneic rejection at the local transplant site.

[0157] In some embodiments, the cloak cells described herein survive in the host without stimulating a host immune response for 1 week or more (e.g., 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years or more, e.g., over the lifespan of the cell and / or its progeny). The cells maintain expression of the cloaking transgene for as long as they survive in the host (e.g., when the cloaking transgene is no longer expressed, the cloak cells can be eliminated by the host's immune system). In some embodiments, the cloak cells further express a transgene encoding a protein (e.g., a fluorescent protein such as GFP or other detectable marker) that allows the cloak cells to be detected in vivo.

[0158] It is contemplated herein that a combination of cloaked and fail-safe cells, whether generated before or after transplantation of the cells into a host, may be useful in cell-based therapies where it may be desirable to eliminate cells that exhibit undesirable proliferation rates while avoiding allogeneic rejection at the local transplant site. The combination of cloaking and fail-safe technologies allows for local immune protection while addressing the risk of the recipient developing malignancies, since the cells provide local immune suppression.

[0159] Method for producing cloak cells The compositions and methods described herein can be used to reduce rejection of allogeneic cells through expression of a cloaking transgene. A wide range of methods have been established for the delivery of proteins to mammalian cells and for the stable expression of genes encoding proteins in mammalian cells, which can be used to produce the cloaked cells described herein.

[0160] Polynucleotides encoding cloaking proteins or therapeutic agents One platform that can be used to achieve therapeutically effective expression of a cloaking protein or therapeutic agent in mammalian cells is via stable expression of a gene encoding the cloaking protein or therapeutic agent (e.g., by integration into the nuclear or mitochondrial genome of the mammalian cell or by episomal concatemer formation in the nucleus of the mammalian cell). A gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein. To introduce an exogenous gene into mammalian cells, the gene can be incorporated into a vector. Vectors can be introduced into cells 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 detail, for example, by 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.

[0161] A cloaking protein or therapeutic agent can also be introduced into mammalian cells by targeting a vector containing a portion of a gene encoding the cloaking 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 affinity for all cell membrane phospholipids). Such constructs can be produced using methods well known to those skilled in the art.

[0162] Recognition and binding of a polynucleotide encoding a cloaking protein or therapeutic agent by mammalian RNA polymerase is important for gene expression. Such sequence elements may include sequence elements within the polynucleotide that exhibit high affinity for transcription factors that recruit RNA polymerase and promote the assembly of a transcription complex at the transcription start site. Such sequence elements include, for example, mammalian promoters, whose sequences can be recognized and bound by specific transcription initiation factors and ultimately by RNA polymerase.

[0163] Polynucleotides suitable for use in the compositions and methods described herein also include polynucleotides encoding cloaking proteins or therapeutic agents downstream of a mammalian promoter. Promoters useful for expressing cloaking proteins or therapeutic agents in mammalian cells include constitutive promoters. Constitutive promoters include the CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, and PGK promoter. Alternatively, promoters derived from viral genomes can be used for stable expression of these agents in mammalian cells. Examples of functional viral promoters that can be used to drive mammalian expression of these agents include the adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter.

[0164] Once a polynucleotide encoding a cloaking protein or therapeutic agent described herein below has been integrated into the nuclear DNA of a mammalian cell, transcription of the polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing the mammalian cell to an external chemical reagent, such as an agent that modulates the binding of transcription factors and / or RNA polymerase to a mammalian promoter, thereby regulating gene expression. The chemical reagent may serve to facilitate the binding of RNA polymerase and / or transcription factors to the mammalian promoter, for example, by removing a promoter-bound repressor protein. Alternatively, the chemical reagent may serve to enhance the affinity of the mammalian promoter for RNA polymerase and / or transcription factors, such that the transcription rate of genes located downstream of the promoter increases in the presence of the chemical reagent. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells to promote gene expression according to established protocols.

[0165] Other DNA sequence elements that can be included in nucleic acid vectors for use in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in polynucleotides, including genes of interest, such that the DNA adopts a three-dimensional orientation favorable for the binding of transcription factors and RNA polymerase at the transcription start site. Thus, polynucleotides for use in the compositions and methods described herein, including polynucleotides encoding cloaking proteins or therapeutic agents, can further include mammalian enhancer sequences. Many enhancer sequences are currently known from mammalian genes, including enhancers from genes encoding mammalian globin, elastase, albumin, α-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those derived from the genetic material of viruses capable of infecting eukaryotic cells. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that direct the activation of eukaryotic gene transcription are disclosed in Yaniv et al., Nature 297:17 (1982). The enhancer can be spliced ​​into a vector containing a polynucleotide encoding a cloaking 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 is in turn located 5' to the polynucleotide encoding the cloaking protein or therapeutic agent.

[0166] The nucleic acid vectors described herein can include a Woodchuck post-transcriptional regulatory element (WPRE). WPREs act at the transcriptional level by promoting nuclear export of transcripts and / or increasing the efficiency of polyadenylation of nascent transcripts, thus increasing the total amount of mRNA in the cell. Addition of a WPRE to a vector can result in substantial improvements in the level of transgene expression from several different promoters both in vitro and in vivo.

[0167] In some embodiments, nucleic acid vectors for use in the compositions and methods described herein contain reporter sequences that can be useful, for example, in verifying gene expression in specific cells and tissues. Reporter sequences that can be provided in a transgene include DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art. When associated with regulatory elements that drive their expression, reporter sequences provide a signal that can be detected by conventional means, including enzymatic, radiographic, colorimetric, fluorescent, or other spectroscopic assays, fluorescence-activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and immunohistochemistry. For example, if the marker sequence is a LacZ gene, the presence of a vector carrying the signal is detected by assaying for β-galactosidase activity. If the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be measured visually by color or light production in a luminometer.

[0168] Techniques for introducing transgenes into cells Transfection Techniques that can be used to introduce transgenes, such as the cloaking transgenes or therapeutic transgenes 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 cells of interest. Mammalian cells, such as human cells, exposed in this manner to an external electric field are then predisposed to uptake of 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. A similar technique, Nucleofection™, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection™ and protocols useful for carrying out this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005) and U.S. Patent Application Publication No. 2010 / 0317114, the disclosures of each of which are incorporated herein by reference.

[0169] Another technique useful for transfection of target cells includes squeezeporation. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to applied stress. This technique is advantageous in that no vector is required for the delivery of nucleic acid to cells, such as human target cells. Squeezeporation is described in detail, for example, in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

[0170] Lipofection represents another technique useful for transfection of target cells. This method involves loading nucleic acids into liposomes, which often present cationic functional groups (e.g., quaternary or protonated amines) toward the exterior of the liposome. This promotes electrostatic interactions between the liposome and the cell due to the anionic nature of the cell membrane, which ultimately leads to the uptake of the exogenous nucleic acid, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. A similar technique that utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acids involves contacting cells with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with polynucleotides to impart a positive charge favorable for interaction with cell membranes include activated dendrimers (e.g., as described 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 guide the uptake of nucleic acids. This technique is described in detail, for example, in U.S. Patent Application Publication No. 2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0171] Another useful tool for inducing the uptake of exogenous nucleic acid by target cells is laser transfection, also called optical transfection, which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize cells and allow polynucleotides to penetrate the cell membrane.The biological activity of this technique is similar to that of electroporation, and in some cases is found to be superior to that of electroporation.

[0172] Impalefection is another technique that can be used to deliver genetic material to target cells. Impalefection relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of a substrate. DNA containing the gene intended for intracellular delivery is attached to the nanostructure surface. A chip with an array of these needles is then pressed against cells or tissue. Cells intercepted by the nanostructures can express the delivered gene. An example of this technique is described in Shalek et al., PNAS 107:1870 (2010), the disclosure of which is incorporated herein by reference.

[0173] Magnetofection can also be used to deliver nucleic acids to target cells. The principle of magnetofection is to associate nucleic acids with cationic magnetic nanoparticles. The magnetic nanoparticles are made from fully biodegradable iron oxide and coated with specific cationic molecules that vary depending on 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 target cells by the influence of an external magnetic field generated by a magnet. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

[0174] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, which involves the use of sound (typically ultrasonic frequencies) to modify the permeability of cell plasma membranes, making cells permeable and allowing polynucleotides to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

[0175] Microvesicles represent another potential vehicle 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 the glycoprotein VSV-G and a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into cells and then catalyze site-specific cleavage of endogenous polynucleotide sequences to prepare the genome of the cell for covalent integration of a polynucleotide of interest, such as a gene or regulatory sequence. The use of such vesicles, also known as gesicles, for genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [Abstract]: Methylation changes in early embryonic genes in cancer [Abstract]: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.

[0176] Viral infection In addition to achieving high transcription and translation rates, stable expression of exogenous genes in mammalian cells can be achieved by integrating a polynucleotide containing the gene into the nuclear genome of the mammalian cell. Various 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, for example, in International Publication No. WO 1994 / 011026, which is incorporated herein by reference. Expression vectors for use in the compositions and methods described herein contain a cloaking or therapeutic transgene and additional sequence elements used, for example, for the expression of these agents and / or the integration of these polynucleotide sequences into the genome of mammalian cells. Particular vectors that can be used for the expression of a cloaking or therapeutic transgene include plasmids containing regulatory sequences, such as promoter and enhancer regions, that direct gene transcription. Other useful vectors for the expression of a cloaking or therapeutic transgene contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0177] Viral vectors for nucleic acid delivery Viral genomes provide a rich source of vectors that can be used for the efficient delivery of genes of interest into the genome of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for gene delivery because polynucleotides contained within such genomes are typically integrated into the nuclear genome of mammalian cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require added proteins or reagents to induce gene integration. Examples of viral vectors include negative-strand RNA viruses such as retroviruses (e.g., Retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, 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, oncoretrovirus, HTLV-BLV complex, lentivirus, alpharetrovirus, gammaretrovirus, and spumavirus (Coffin, J.M., Retroviridae: The viruses and their replication, Virology, Third 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 simian virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which is incorporated herein by reference as it relates to viral vectors for use in gene therapy.

[0178] AAV vectors for nucleic acid delivery In some embodiments, the cloaking or therapeutic transgenes described herein are incorporated into rAAV vectors and / or virions to facilitate their introduction into cells. rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs containing (1) a promoter, (2) a heterologous sequence to be expressed (e.g., a cloaking or therapeutic transgene described herein), and (3) viral sequences that facilitate the integration and expression of the heterologous gene. The viral sequences may include AAV sequences required in cis for replication and packaging of DNA into virions (e.g., functional ITRs). Such rAAV vectors may also contain a marker or reporter gene. Useful rAAV vectors have one or more AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype suitable for a particular application. This method for using rAAV vectors is 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.

[0179] The transgenes and vectors described herein (e.g., promoters operably linked to cloaking 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 outer, non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins, VP1, VP2, and VP3, required for virion assembly. Construction of rAAV virions is described, for example, in U.S. Pat. Nos. 5,173,414; 5,139,941; 5,863,541; 5,869,305; 6,057,152; and 6,376,237; as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the disclosures of each of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0180] rAAV virions useful in conjunction with the compositions and methods described herein include those derived from various 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.

[0181] Pseudotyped rAAV vectors are also useful in connection with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) that are pseudotyped with a capsid gene from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques for constructing and using 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).

[0182] AAV virions with mutations in the virion capsid can be used to infect specific cell types more efficiently than non-mutated capsid virions. For example, suitable AAV mutants can have ligand insertion mutations to facilitate targeting of AAV to 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 capsid hybrids generated by molecular propagation of virus and by exon shuffling. See, for example, Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0183] Genome editing In addition to the above, various tools have been developed that can be used to integrate a gene of interest into a target cell, such as a mammalian cell. One such method that can be used to integrate a polynucleotide encoding a target gene into a target cell involves the use of a transposon. A transposon is a polynucleotide that encodes a transposase enzyme and contains a polynucleotide sequence or gene of interest flanked by 5' and 3' excision sites. Once the transposon is delivered into a cell, expression of the transposase gene begins, resulting in an active enzyme that cleaves the gene of interest from the transposon. This activity is mediated by site-specific recognition of the transposon excision site by the transposase. In some instances, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of the mammalian cell by transposase-catalyzed cleavage of similar excision sites present in the cell's nuclear genome. This allows the gene of interest to be inserted into the nuclear DNA that has been cut at the complementary excision site, and then the covalent ligation of phosphodiester bonds that connects the gene of interest to the DNA of mammalian cell genome completes the integration process.In certain cases, transposon can be a retrotransposon, such that the gene encoding the target gene is first transcribed into RNA product, and then reverse transcribed into DNA before being integrated into mammalian cell genome.Exemplary transposon systems are piggyback transposon (for example, as described in detail in International Publication No. 2010 / 085699) and sleeping beauty transposon (for example, as described in detail in US Patent No. 2005 / 0112764), the disclosures of which relate to the transposon used in gene delivery to target cells and are incorporated herein by reference.

[0184] Another tool for target gene integration into the genome of a target cell is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, which first evolved in bacteria and archaea as an adaptive defense mechanism against viral infection. The CRISPR / Cas system contains palindromic repeat sequences within plasmid DNA and the associated Cas9 nuclease. This ensemble of DNA and proteins first integrates foreign DNA into the CRISPR locus, thereby directing site-specific DNA cleavage of the target sequence. Polynucleotides containing these foreign sequences and the repeated spacer elements of the CRISPR locus are then transcribed in the host cell to generate guide RNAs, which can then anneal to the target sequence and localize the Cas9 nuclease to the site. In this way, highly site-specific Cas9-mediated DNA cleavage can be induced in the foreign polynucleotide, because the interaction that brings Cas9 into close proximity to the target DNA molecule is governed by RNA:DNA hybridization. As a result, CRISPR / Cas systems can be designed to cleave any target DNA molecule of interest. This technology has been utilized to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and can be used as an efficient means of site-specifically editing target cell genomes to cleave DNA prior to integration of a gene encoding a target gene. The use of CRISPR / Cas to regulate gene expression is described, for example, in U.S. Patent No. 8,697,359, the disclosure of which is incorporated herein by reference as it relates to the use of the CRISPR / Cas system for genome editing. Alternative methods for site-specific cleavage of genomic DNA prior to integration of a gene of interest into a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike CRISPR / Cas systems, these enzymes do not contain a guided polynucleotide to localize to a specific target sequence. Instead, target specificity is controlled by a DNA-binding domain 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 by reference herein as they relate to compositions and methods for genome editing.

[0185] Additional genome editing techniques that can be used to integrate a polynucleotide encoding a target gene into the genome of a target cell include the use of ARCUS™ meganucleases, which can be rationally designed to site-specifically cleave genomic DNA. The use of these enzymes to integrate a gene encoding a target gene into the genome of a mammalian cell is advantageous in view of the defined structure-activity relationship established for such enzymes. Single-stranded meganucleases can be modified at specific amino acid positions to create nucleases that selectively cleave DNA at desired locations and enable site-specific integration of the target gene into the nuclear DNA of the target cell. These single-stranded nucleases are broadly described, for example, in 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.

[0186] Cloaking transgene expression The cloaking transgenes described herein (e.g., one or any combination of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)) are expressed in an amount sufficient to produce a cloaking effect (e.g., in an amount sufficient to prevent rejection when injected into a subject (e.g., a mammalian subject such as a mouse, rat, or human)). Transgene expression can be considered to produce a cloaking effect if subcutaneous injection of cloaked cells produces a teratoma that is not cleared by the subject's immune system. The cloaking transgene is also expressed at a level sufficient to promote production of the protein encoded by the transgene. Protein production can be detected using routine methods known to those of skill in the art (e.g., immunohistochemistry, Western blot analysis, or other methods that allow visualization or protein expression). Preferably, expression of the cloaking transgene is such that all eight proteins encoded by the cloaking transgene (PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6) can be detected in cloaked cells (e.g., as detected by immunohistochemistry using antibodies against proteins encoded by the cloaking transgene).

[0187] In some embodiments, the cloaking transgene is expressed in cloaked cells at a level similar to the level of endogenous gene expression in activated leukocytes such as T cells (e.g., expression in activated leukocytes from the same species, such as activated leukocytes isolated from lymphoid organs, e.g., cloaked mouse cells, is similar to expression in activated leukocytes isolated from mouse lymphoid organs). Expression of one or more cloaking transgenes (e.g., one, two, three, four, five, six, seven, or eight of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)) is equal to or greater than the expression of the endogenous gene in activated leukocytes (e.g., T cells) from the same species (e.g., the expression level of the cloaking 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, or more times greater than the expression level of the endogenous gene in activated leukocytes). In some embodiments, all eight cloaking transgenes are expressed at levels equal to or greater than the expression level of the endogenous gene in activated leukocytes from the same species. Activated leukocytes can be isolated from lymphoid organs, or leukocytes such as T cells can be activated in vitro using anti-CD3 / CD28 beads or other methods used 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 cloak cells can also be compared to gene expression levels reported in activated T cell profiling studies (see, e.g., Palacios et al., PLOSone 2:e1222 (2007)). In some embodiments, cloaking transgene expression is compared to expression of the endogenous gene in a wild-type version of the cell (e.g., a stem cell, e.g., an embryonic stem cell from the same species as the cloak cell).Expression of one or more cloaking transgenes (e.g., one, two, three, four, five, six, seven, or eight 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 or more times higher in the cloaked cells compared to expression of the endogenous gene(s) in an unmodified wild-type cell of the same cell type (e.g., a stem cell, e.g., an embryonic stem cell from the same species) as the cloaked cells. In some embodiments, all eight cloaking transgenes are expressed at levels higher (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100 times higher) than the expression level of the endogenous gene in a wild-type version of the cell (e.g., a stem cell, e.g., an embryonic stem cell from the same species as the cloaked cell). Gene expression can be assessed by direct comparison with 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)).

[0188] Methods for providing local immunosuppression at the transplant site - Patents.com Also featured are methods for providing local immunosuppression at the transplant site.

[0189] The method includes providing a cell and expressing a set of transgenes in the cell, each transgene encoding a cytoplasmic, membrane-bound, or locally acting gene product whose function is to mitigate the function of graft-attacking leukocytes and NK cell activation or to act as a defense mechanism against leukocyte attack.

[0190] The set 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 Serpin B9 (Spi6). In an embodiment, the set of transgene genes includes PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6.

[0191] Optionally, the method further includes 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 embodiments, the TGF-β or biological agent is locally acting.

[0192] Techniques for introducing various genetic modifications, such as transgenes, into animal cells are described herein and are generally known in the art.

[0193] In embodiments of this method, the cells are stem cells, cells amenable to genome editing, and / or sources of therapeutic cell types (e.g., cells that can be differentiated into lineage-restricted cells for cell therapy or cells of a desired target tissue). In embodiments, the cells are embryonic stem cells, induced pluripotent stem cells, adult stem cells, tissue-specific stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germline stem cells, lung stem cells or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, or neural stem cells or progenitor cells. In some embodiments, the cells are 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 cells are fibroblasts, epithelial cells, or endothelial cells. The cells can be vertebrate cells, e.g., mammalian cells, such as human or mouse cells. In some embodiments, 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 within the tissue or organ to be transplanted. In some embodiments, cloak cells (e.g., cloak stem cells) are differentiated in vitro into the tissue or organ for transplantation using methods known to those of skill in the art.

[0194] In some embodiments, between 1 million and 100 billion cloaked cells (e.g., 1 x 10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7, 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 or 1 x 10 11 The cloak cells are administered to the subject at or near the transplant site or into the organ or tissue to be transplanted.

[0195] Techniques for transplanting genetically modified cells into the transplant site of an allogeneic host are described herein and are known in the art.

[0196] Expression of therapeutic agents by cloak cells The cloak cells described herein can be further modified to express a therapeutic agent. In some embodiments, the therapeutic agent is a protein. The therapeutic protein can be a wild-type form of a protein that is deficient in a subject, such as a protein that is mutated or produced in insufficient amounts (e.g., produced at low levels or not produced) by the subject's cells. In some embodiments, the therapeutic protein is an inhibitory antibody (e.g., an antibody that blocks or neutralizes protein function). The cloak cells can be modified to produce an inhibitory antibody to treat a subject having or at risk of developing a disease or condition associated with overproduction or aberrant production of a protein (e.g., production by cells that do not normally produce the protein, production of a protein at a time or location where the protein is not normally produced, or production of an excessive amount of a protein). In some embodiments, the therapeutic antibody is an agonist antibody (e.g., an activating antibody). Agonist antibodies can act by binding to and activating endogenous receptors (e.g., inducing or increasing signaling downstream of receptor activation or changing the conformation of the endogenous receptor to an open or active state). Cloak cells can be modified to produce agonist antibodies to treat subjects having or at risk of developing a disease or condition associated with underactivation of a receptor or signaling pathway. Cloak cells can be modified to produce a therapeutic protein or antibody using the methods described herein or other methods known to those of skill in the art. Cloak cells producing secreted proteins or antibodies can be delivered as circulating cells, injected into a tissue, organ, or body site in need of a therapeutic protein or antibody, or injected subcutaneously to produce cloaked subcutaneous tissue. Cloak cells producing transmembrane or membrane-bound proteins can be injected at or near the site of endogenous cells that respond to the therapeutic protein.

[0197] In some embodiments, the cloak cells described herein provide wild-type copies of genes that are mutated in a subject (e.g., the cloak cells are "wild-type cells" that do not have the genetic cause of the disease and express 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). Such cells can be used to treat subjects with diseases or conditions caused by mutations in endogenous genes (e.g., subjects with metabolic disorders associated with one or more mutations described herein below).

[0198] A list of exemplary therapeutic agents that may be administered with or produced by cloak cells and associated diseases or conditions that may be treated using these therapeutic agents is provided in Table 2 below.

[0199] [Table 2]

[0200] [Table 3]

[0201] [Table 4]

[0202] [Table 5]

[0203] [Table 6]

[0204] [Table 7]

[0205] [Table 8]

[0206] Inducible systems for the expression of therapeutic agents When continuous administration of a therapeutic agent expressed by cloak cells is required to treat a disease or condition, the therapeutic agent can be expressed using a constitutive promoter (e.g., CAG, CMV, or another constitutive promoter) described herein or known to those of skill in the art. When a therapeutic agent is needed intermittently (e.g., during periods of recurrence or flare-ups that occur during a disease or condition, but not when the subject is asymptomatic), the therapeutic agent can be expressed by an inducible promoter, providing the ability to express the therapeutic agent only when needed. One exemplary class of therapeutic agent that can be delivered using an inducible promoter is TNFα inhibitors. TNFα inhibitors are currently used to treat rheumatoid arthritis, but are only administered intermittently during flares of joint inflammation because constitutive administration of TNFα can result in systemic immunosuppression. If cloak cells are modified to express a TNFα inhibitor under the control of an inducible promoter, the cloak cells can be used to deliver TNFα intermittently, thus eliminating the need for repeated injections. Other therapeutic agents that have potentially deleterious effects when administered continuously can also be expressed intermittently using inducible promoters as described herein. Exemplary inducible expression systems are described below.

[0207] Tetracycline Response Element One 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 a gene of interest (e.g., a therapeutic transgene to be expressed by cloak cells), typically with a minimal promoter that has very low basal expression. A protein called rtTA, which also needs to be expressed by cloak 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 gene of interest is no longer expressed. An advanced version of this system, the Tet-On Advanced Transactivator (rtTA2), is available. 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.

[0208] light guidance system Another method for inducible activation of gene expression involves the use of optogenetics, which uses light-sensitive proteins to manipulate gene expression. Recent developments in optogenetics that can be used to inducibly express therapeutic agents in cloak cells include a class of proteins that undergo conformational changes and dimerization in response to blue light. These proteins are fused to DNA-binding and transcriptional components that have been shown to bind to specific promoter sequences and activate transcription when combined with exposure to blue light (Wang et al., Nat Methods, 9:266-269, 2012). Because blue light can be applied to the skin near cloaked subcutaneous tissue to induce the production of therapeutic agents by cloak cells, methods for inducibly activating gene expression can be used to control the production of therapeutic agents in subcutaneously administered cloak cells.

[0209] Radiogenetics A third method for inducibly activating gene expression (e.g., expression of a therapeutic agent by cloak cells) involves the use of radio waves. In one version of the radio wave-inducible expression system, the TRPV1 receptor is fused to a GFP-binding domain and coexpressed with a form of ferritin linked 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, allowing calcium influx, which activates the transcription factor NFAT. Therapeutic agents can be inducibly expressed using this system when operably linked to an NFAT-sensitive promoter element (e.g., SRE-CRE-NFATRE) and coexpressed with TRPV1-GFP and GFP-ferritin. Radio wave-inducible expression offers the advantage that radio waves can penetrate tissues, allowing expression to be induced in cells far from the body. For example, radiogenetics can be used to regulate gene expression in the retina. Thus, the present method can be used to inducibly express therapeutic transgenes in cloak cells using non-invasive and non-harmful radio waves.

[0210] Destabilizing domain system Gene expression can also be regulated using a destabilization domain system. A transgene encoding a protein of interest (e.g., a therapeutic agent described herein) can also contain a destabilization domain, such that the resulting protein product contains the protein of interest fused to the destabilization domain. Exemplary destabilization domains include mutants of human FK506 and rapamycin-binding protein (FKBP12), which confer instability to proteins to which they are fused. 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 FKBP12 destabilization domains. The destabilization domain promotes protein degradation. If expression of a protein of interest (e.g., a therapeutic agent) is desired, small molecule synthetic ligands can be used to stabilize destabilization domain-containing proteins. The small molecule ligand Shield-1 (Shld1) can be used to stabilize FKBP12 mutant-containing proteins by protecting them from degradation. Other destabilization domains that can be used to regulate the expression of a protein of interest include mutants of Escherichia coli (E. coli) dihydrofolate reductase (ecDHFR) and mutants of the human estrogen receptor ligand binding domain (ERLBD), which confer instability that results in degradation when fused to a protein of interest 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 each of which are incorporated herein by reference as they relate to destabilization domain systems).

[0211] Cmate switch induction system Another method for inducible activation of gene expression involves the use of the cumate gene switch system. In this system's repressor configuration, regulation is mediated by binding of a repressor (CymR) to an operator site (CuO) placed downstream of a strong constitutive promoter. Addition of the small molecule cumate relieves repression, allowing transgene expression. Alternatively, a reverse cumate transactivator (rcTA) can be inserted upstream of a minimal CMV promoter operably linked to a transgene encoding a therapeutic agent. Six repeats of the cumate operator (6xCuO) can be inserted immediately before the translation start (ATG) of the therapeutic transgene. In the absence of cumate, rcTA cannot bind to 6xCuO, and therefore the transgene encoding the therapeutic agent is not transcribed because 6xCuO is inactive. When cumate is added, it forms a complex with rcTA, which allows binding to 6xCuO and transcription of the transgene encoding the therapeutic agent (Mullick et al., 2006).

[0212] Ecdysone induction system Another example of an inducible gene expression system is the ecdysone-inducible system, in which an N-terminal truncation of the ecdysone receptor (EcR) fused to the activation domains of retinoid X receptor (RXR) and Vp16 (VpEcR) is inserted into the 5' untranslated region of a gene expressed by cloak cells so that it is coexpressed by the endogenous promoter. An ecdysone response element (EcRE) with a downstream minimal promoter can be inserted directly upstream of the start codon of a transgene encoding a therapeutic agent. Coexpressed 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 the EcRE, and thus the transgene encoding the therapeutic agent is not transcribed. In the presence of ecdysone or muristerone A, the dimerized RXR / VpEcR can bind to the EcRE, allowing transcription of the transgene encoding the therapeutic agent (No et al., 1996). Because ecdysone administration has no apparent effects on mammals, its use to regulate genes should be superior to the transient inducible expression of any gene.

[0213] Ligand reversible dimerization system In another example, a transgene encoding a therapeutic agent can be modified to be functionally divided into portions / domains, such as a 5' portion and a 3' portion, and an FKBP peptide sequence can be inserted into each domain. An IRES (internal ribosome entry site) sequence can be placed between the two domains, allowing the two distinct domains to be co-transcribed to generate two separate proteins. In the absence of a dimerizer, the two separate domains of the therapeutic agent are functionally inactive. Upon introduction of a dimerizer, such as rapamycin or AP20187, the FKBP peptide dimerizes, bringing the 5' and 3' domains of the therapeutic agent together and reconstituting an active protein (Rollins et al., 2000).

[0214] Cell-Based Delivery of Therapeutic Agents Treatment of age-related macular degeneration or retinal dystrophy In one example, cloak cells can be modified to produce a VEGF inhibitor, such as a VEGF trap (e.g., a soluble decoy receptor, e.g., aflibercept, described in Holash et al., Proc Natl Acad Sci USA 99:11383-11398, 2002, incorporated herein by reference), to treat age-related macular degeneration (AMD) or retinal dystrophy. VEGF traps are biological substances that bind to and inhibit VEGF, an angiogenic protein that can promote the formation of abnormal blood vessels. VEGF traps are used to treat wet AMD, which is characterized by the 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 periodic intraocular injection. Cloak cells can be modified to produce a VEGF trap or another VEGF inhibitor by expressing a transgene encoding the VEGF trap or another VEGF inhibitor operably linked to a constitutive or inducible promoter. Cloaked cells (e.g., stem cells) expressing a VEGF inhibitor (e.g., a VEGF trap) can be differentiated into retinal pigment epithelial (RPE) cells before administration to the eye using methods known to those skilled in the art, or isolated RPE cells can be modified to express a cloaking transgene and a VEGF inhibitor. To treat wet AMD or retinal dystrophy, 25,000 to 100,000 cloaked RPE cells (e.g., 25,000, 50,000, 75,000, or 100,000 cloaked RPE cells) expressing a VEGF inhibitor (e.g., a VEGF trap) can be injected into the subretinal space of each eye. Other VEGF inhibitors suitable for use in the compositions and methods described herein include soluble forms of VEGF receptors (e.g., soluble VEGFR-1 or NRP-1), platelet factor-4, prolactin, SPARC, and VEGF-inhibiting antibodies (e.g., bevacizumab and ranibizumab).

[0215] Treatment of Parkinson's disease In another example, cloaked 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 suffering from Parkinson's disease, which is characterized by loss of dopaminergic neurons. 25,000 to 100,000 cloaked dopaminergic neurons (e.g., 25,000, 50,000, 75,000, or 100,000 cloaked dopaminergic neurons) can be administered to the brain of a subject suffering from Parkinson's disease (e.g., stereotactically injected into the substantia nigra).

[0216] Treatment of myocardial infarction The cloak cells described herein can also be used to treat myocardial infarction (e.g., myocardial infarction, commonly known as a heart attack). Myocardial infarction occurs when blood flow to a portion of the heart is reduced or stopped, causing damage to the heart muscle. To treat a subject suffering from myocardial infarction, cloak cells (e.g., stem cells) can be differentiated into cardiomyocytes using methods known by those skilled in the art, or isolated cardiomyocytes can be modified to express a cloaking transgene. 500 to 5 billion cloaked cardiomyocytes (e.g., 5 x 10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 or 5 x 10 9 The cloaked cardiomyocytes can be administered to a subject by injection into the myocardium to treat a subject suffering from a myocardial infarction (e.g., to replace dead or damaged cardiomyocytes).

[0217] Treatment of osteoarthritis and rheumatoid arthritis In another example, the cloak cells described herein can be used to treat osteoarthritis or rheumatoid arthritis. Osteoarthritis and rheumatoid arthritis (RA) are characterized by joint inflammation and are commonly treated with anti-inflammatory therapeutic agents. To treat a subject suffering from osteoarthritis or RA, cloak cells can be modified to express an anti-inflammatory biological substance (e.g., a TNFα inhibitor (e.g., a TNFα-inhibiting antibody)), which is already used clinically for the treatment of RA. Cloak cells can be modified to produce an anti-inflammatory biological substance, such as a TNFα inhibitor, by expressing a transgene encoding the anti-inflammatory biological substance operably linked to a constitutive or inducible promoter. Cloak cells (e.g., stem cells) expressing an anti-inflammatory biological substance (e.g., a TNFα inhibitor) can be differentiated into articular fibroblasts before administration to a joint using methods known to those skilled in the art, or isolated articular fibroblasts can be modified to express a cloaking transgene and an anti-inflammatory biological substance. 1 million to 100 million cloaked articular fibroblasts (e.g., 1 x 10) expressing anti-inflammatory biological substances 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 or 1 x 10 8Cloak cells (articular fibroblasts) can be injected into arthritic or inflamed joints (depending on the size of the joint) to treat osteoarthritis or RA. Anti-inflammatory biological substances that can be expressed by cloak cells to treat osteoarthritis or RA include TNFα inhibitors (adalimumab, etanercept, infliximab, golimumab, certolizumab), interleukin-6 (IL6) receptor inhibitors (e.g., tocilizumab), IL1 receptor inhibitors (e.g., anakinra), or other drugs used to treat RA (e.g., abatacept, rituximab).

[0218] Diabetes Treatment Cloak cells can be used to treat diabetes (e.g., type 1 or type 2 diabetes). Type 1 diabetes results from the inability of the pancreas to produce sufficient insulin. Type 2 diabetes begins with insulin resistance, but as the disease progresses, insulin deficiency can develop. To treat a subject with diabetes, cloak cells can be modified to express insulin, or insulin-expressing cells from a healthy subject (e.g., pancreatic beta cells from a subject without diabetes) can be modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the cloaking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject with diabetes. Cloak cells can be modified to produce insulin by expression of an insulin-encoding transgene operably linked to a constitutive or inducible promoter. Insulin-expressing cloak cells (e.g., stem cells) can be differentiated into insulin-producing cells (e.g., pancreatic β cells) using methods known to those of skill in the art, or can be administered without differentiation, or isolated pancreatic β cells can be modified to express a cloaking transgene and, optionally, an insulin-encoding transgene. Approximately 800 million to 3 billion cloaked pancreatic β cells (e.g., 8×10) that express insulin (e.g., endogenously express insulin or express insulin due to expression of a transgene encoding insulin) can be administered. 8 , 9×10 8 , 1×10 9 , 2 × 10 9 or 3 x 10 9 The cloaked pancreatic beta cells can be subcutaneously injected into a subject to create insulin-producing subcutaneous tissue to treat diabetes.

[0219] Treatment of Hemophilia In another example, the cloak cells described herein can be used to treat hemophilia. Patients with hemophilia do not produce functional factor VIII protein, a critical blood component required for blood clotting. These patients may experience severe bleeding, and standard care involves multiple weekly infusions of purified factor VIII protein. To treat subjects with hemophilia, cloak cells can be modified to express an additional transgene encoding factor VIII. Factor VIII is constitutively expressed in cloak cells by operably linking it to a constitutive promoter such as CMV or CAG. Factor VIII-expressing cloak cells (e.g., stem cells) can be differentiated into cells that produce blood clotting factors (e.g., hepatic sinusoidal cells or endothelial cells) using methods known to those of skill in the art before administration, or can be administered without differentiation, or factor VIII-expressing hepatic sinusoidal cells or endothelial cells isolated from a healthy subject (e.g., a subject without hemophilia) can be modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the cloaking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject with hemophilia. Isolated factor VIII-expressing hepatic sinusoidal or endothelial cells from a healthy subject that have been modified to express one or more cloaking transgenes can be further modified to express a transgene encoding factor VIII, if necessary, to ensure high levels of factor VIII expression. Factor VIII-expressing (e.g., endogenously expressing factor VIII or expressing factor VIII due to expression of a transgene encoding factor VIII) cells (e.g., 8 x 10 cells) are used. 8 , 9×10 8 , 1×10 9 , 2 × 10 9 or 3 x 10 9The cloaked cells can be subcutaneously injected into a subject to create a cloaked subcutaneous tissue that produces factor VIII to treat hemophilia.

[0220] Treatment of metabolic disorders The cloak cells of the present invention can also be used to treat inherited metabolic disorders, in which most inherited metabolic disorders involve a single enzyme that is not produced in the body or is produced in a defective form. Inherited metabolic disorders include lysosomal storage disorders such as Hurler syndrome (α-L-iduronidase deficiency), Niemann-Pick disease (SMPD1, NPC1, or NPC2 mutations), Tay-Sachs disease (HEXA mutations), Gaucher disease (GBA gene mutations), Fabry disease (α-galactosidase deficiency due to GLA mutations) and Krabbe disease (galactosylceramidase deficiency due to GALC mutations); galactosemia (galactosinase or galactose-1-phosphate urea transferase deficiency), maple syrup urine disease (enzyme BCKD deficiency); phenylketonuria (enzyme PAH deficiency); glycogen storage diseases (GSDs) such as GSD0 (glycogen synthase (GYS2) deficiency), GSD1 / von Gierke disease (glucose-6-phosphatase (G6PC) deficiency), GSD2 / Pompe disease (acid α-glucosidase (GAA) deficiency), GSD3 / GSD4 / GSD5 / GSD6 / GSD7 / GSD8 / GSD9 / GSD10 / GSD11 / GSD12 / GSD13 / GSD14 / GSD15 / GSD16 / GSD17 / GSD18 / GSD19 / GSD20 / GSD21 / GSD22 / GSD23 / GSD24 / GSD25 / GSD26 / GSD27 / GSD28 / GSD29 / GSD30 / GSD31 / GSD32 / GSD33 / GSD34 / GSD35 / GSD36 / GSD37 / GSD38 / GSD40 / GSD41 / GSD42 / GSD43 / GSD44 / GSD55 / GSD56 / GSD57 / GSD58 / GSD60 / GSD6 GSD 3 / Cori's disease or Forbes' disease (glycogen debranching enzyme AGL deficiency), GSD 4 / Anderson's disease (glycogen branching enzyme (GBE1) deficiency), GSD 5 / McArdle's disease (muscle glycogen phosphorylase (PYGM) deficiency), GSD 6 / Haas's 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 (deficiency of β-enolase (ENO3)) or GSD 15 (deficiency of glycogenin-1 (GYG1));Mitochondrial diseases, such as mitochondrial myopathy (Kerns-Sayre syndrome (KSS), due to mitochondrial DNA deletions) and chronic progressive external ophthalmoplegia (CPEO, due to mitochondrial DNA deletions or duplications or ANT1, POLG, POLG2 or PEO1 mutations), diabetes and hearing loss (DAD, due to mitochondrial DNA mutations at position 3243 encoding tRNALeu(UUR)), Leber's hereditary optic neuropathy (LHON, due to MT-ND1, MT-ND4, MT-ND4L and MT-ND6) Leigh syndrome (associated with mutations in SURF1, MT-ATP6, MT-ND2, MT-ND3, MT-ND5, MT-ND6, BCS1L, NDUFA10, SDHA, NDUFS4, NDUFAF2, NDUFA2, NDUFAF6, COX15, NDUFS3, NDUFS8, FOXRED1, NDUFA9, NDUFA12, NDUFS7), neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP, due to mutations in MT-ATP6), myoneurogenic gastrointestinal encephalopathy (MNGIE, due to mutations in TYMP), myoclonic epilepsy with ragged red fibers (MERRF, sin MT-TK, MT-TL1, MT-TH, MT-TS1, MT-TS2 or MT-TF) or mitochondrial myopathies, encephalomyopathies, lactic acidosis, stroke-like symptoms (due to mutations in MELAS, MT-ND1, MT-ND5, MT-TH, MT-TL1 or MT-TV); Friedrich ataxia (mutations in FXN); peroxisomal disorders such as 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's disease (mutations in Wilson's disease protein ATP7B) and hemochromatosis (mutations in the human hemochromatosis protein HFE);Organic acid metabolism disorders, such as methylmalonic acidemia (mutations in MUT, MMAA, MMAB, MMADHC, or MCEE) and propionic acidemia (mutations in PCCA or PCCB); urea cycle disorders, such as ornithine transcarbamylase (OTC) deficiency, arginase (ARG1) deficiency, argininosuccinate lyase (ASL) deficiency, argininosuccinate synthase 1 (ASS1) deficiency, citrin deficiency, carbamoylphosphate synthase 1 (CPSI) deficiency, N-acetylglutamate synthase (NAGS) deficiency, and ornithine translocase (ORNT1) deficiency;

[0221] To treat a subject suffering from a metabolic disorder, cloak cells can be modified to express a transgene encoding the wild-type form of the gene that is mutated in the subject or the enzyme that is deleted or defective in the subject (see Table 2), or cells from a healthy subject (e.g., a subject without a metabolic disorder) that express the wild-type form of the gene that is mutated in the subject or the enzyme that is defective 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 cloaking 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. A transgene encoding the wild-type form of the gene that is mutated in the subject or the enzyme that is deleted or defective in the subject can be constitutively expressed in the cloak cells by being operably linked to a constitutive promoter such as CMV or CAG, or can be inducibly expressed using one of the inducible expression systems described herein. Cloak cells (e.g., stem cells) modified to express the wild-type form of a gene mutated in a subject or the enzyme deleted or defective in the subject can be differentiated into cells that normally express the gene or enzyme before administration using methods known to those of skill in the art, or can be administered without differentiation. Alternatively, cells isolated from a healthy subject that express the wild-type form of a gene or enzyme deleted 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 cloaking 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 yet been diagnosed with a specific mutation prior to treatment, the subject can be evaluated using standard methods to identify the mutated gene associated with the metabolic disorder and ensure that the cloak cells express the corresponding wild-type gene.800 million to 3 billion cloak cells (e.g., 8 x 10) expressing the wild-type form of the gene that is mutated in the subject. 8 , 9×10 8 , 1×10 9 , 2 × 10 9 or 3 x 10 9 The cloaked cells can be injected subcutaneously to create cloaked subcutaneous tissue that produces the corresponding wild-type protein.

[0222] Methods for controlling cloak cell division In one aspect, a method is provided for controlling the proliferation of cells at a transplant site in an allogeneic host (e.g., to reduce the tumorigenic potential of cells at the transplant site or to reduce the proliferation of cells that have become tumorigenic at the transplant site).

[0223] The method includes providing cells genetically modified to contain at least one mechanism for providing local immunosuppression at a transplant site when the cells or a population of such cells are transplanted into an allogeneic host; genetically modifying cell division loci (CDLs) in the cells, wherein the CDLs are one or more loci whose transcription products are expressed by dividing cells (e.g., all dividing cells that contain one or more immunosuppressive transgenes), and the genetic modification of the CDLs is achieved by one or more of the following: a) an ablation link (ALINK) system comprising a DNA sequence encoding a negative selection marker transcriptionally linked to a DNA sequence encoding the CDL; and b) an inducible exogenous activator of regulation of CDLs (EARC) system comprising an inducible activator-based gene expression system operably linked to the CDL. the method includes genetically modifying a cell or a population of cells, including a plurality of cells, either maintaining the genetically modified cells comprising the ALINK system in the absence of an inducer of the negative selection marker to expand the genetically modified cells comprising the ALINK system or exposing the cells comprising the ALINK system to an inducer of the negative selection marker to ablate and / or inhibit the proliferation of the genetically modified cells comprising the ALINK system, and / or exposing the genetically modified cells comprising the EARC system to an inducer of an inducible activator-based gene expression system to expand the genetically modified cells comprising the EARC system or maintaining the cells comprising the EARC system in the absence of an inducer of an inducible activator-based gene expression system to prevent or inhibit the proliferation of the genetically modified cells comprising the EARC system, and transplanting the cell or population of cells into a transplant site in an allogeneic host. Cells modified to control cell division using one or more ALINK and / or EARC systems in one or more CDLs (e.g., two, three, four or more CDLs) may be referred to as "fail-safe cells." The number of cells that can be grown from a single fail-safe cell before the cell loses activity of all systems that control cell division (e.g., ALINK or EARC) due to genetic mutation (e.g., the number of cell divisions required for a cell to "escape" from control and, based on mathematical modeling, indicate uncontrolled cell growth) (clonal volume) determines the fail-safe volume.The amount of failsafe depends on the number of ALINKs and the number of ALINK target CDLs. The failsafe characteristics are further detailed in Table 3.

[0224] [Table 9]

[0225] In various embodiments, the CDL is a locus identified as an "essential gene" as described in Wang et al., 2015 (which is incorporated by reference in its entirety as if expressly set forth herein). The essential genes described in Wang et al., 2015 are identified by calculating a score (i.e., a CRISPR score) for each gene, which score represents the fitness cost imposed by inactivating the gene. In one embodiment, the CDL has a CRISPR score (CS) of less than about -1.0 (Table 5, column 5).

[0226] In various embodiments, the CDL is one or more loci that encode gene products associated with cell division and / or replication (Table 5, column 6). For example, in various embodiments, the CDL is a locus that encodes gene products associated with one or more of i) the cell cycle, ii) DNA replication, iii) RNA transcription and / or protein translation, and iv) metabolism (Table 5, column 7).

[0227] In one embodiment, the CDL is one or more cyclin-dependent kinases involved in regulating cell cycle progression (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, the CDL is one or more cyclins involved in regulating cell cycle progression by activating one or more CDKs, such as 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, the CDL is one or more genetic loci involved in the anaphase-promoting complex, which controls progression from metaphase to anaphase during the M phase of the cell cycle (Peters, 2002). In one embodiment, the CDL is one or more loci involved in kinetochore components that control progression from metaphase to anaphase during the M phase of the cell cycle. In one embodiment, the CDL is one or more loci involved in microtubule components that control microtubule movement required for the cell cycle (Cassimeris, 1999).

[0228] In various embodiments, the CDL is one or more gene loci involved in housekeeping. As used herein, the term "housekeeping gene" or "housekeeping locus" refers to one or more genes necessary for maintaining basic cellular functions. Housekeeping genes are expressed in all cells of an organism under normal and pathophysiological conditions.

[0229] In various embodiments, the CDL is one or more loci encoding gene products related to cell division and / or proliferation, and has a CRISPR score of less than about -1.0. For example, in one embodiment, the CDL is one or more loci encoding gene products related to one or more of: i) cell cycle, ii) DNA replication, iii) RNA transcription and / or protein translation, and iv) metabolism, and has a CRISPR score of less than about -1.0. In one embodiment, the CDL is also a housekeeping gene.

[0230] 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.

[0231] Cells can be modified to be "fail-safe" cells by linking expression of a CDL with expression of a DNA sequence encoding a negative selection marker, thereby allowing drug-induced ablation of mitotically active cells expressing both the CDL and the negative selection marker. Ablation of proliferating cells may be desired, for example, when cell proliferation is uncontrolled and / or accelerated compared to the cell's normal division rate (e.g., uncontrolled cell division exhibited by cancerous cells) or when the therapeutic need for the cells has passed. Ablation of amplified cells can be achieved by genetic modifications to cells, referred to herein as "ablation links" (ALINKs), that link expression of a DNA sequence encoding a negative selection marker with expression of a CDL, thereby allowing elimination or sufficient inhibition (sufficient inhibition that inhibits cell proliferation rates to rates too low to contribute to tumor formation) of ALINK-modified amplified cells expressing the CDL locus. Cells expressing the negative selection marker in the presence of a prodrug or other inducer of the negative selection system will cease proliferation or die, depending on the mechanism of action of the selection marker. The cells can be modified to contain homozygous, heterozygous, hemizygous, or compound heterozygous ALINK. In one embodiment, negative selection markers can be introduced into all functional CDL alleles to improve the fidelity of ablation. In one preferred embodiment, negative selection markers can be introduced into all functional CDL alleles. A fail-safe system can be used to eliminate all cloak cells, if necessary.

[0232] ALINK can be introduced into any position of the CDL, allowing for simultaneous expression of the CDL and a negative selection marker.

[0233] 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.

[0234] DNA encoding a negative selection marker (e.g., HSV-TK) can be inserted into a CDL (e.g., CDK1) in a host cell such that expression of the negative selection marker causes the host cell expressing the negative selection marker and consequently the CDL to die in the presence of an inducer (e.g., a prodrug) of the negative selection marker (e.g., ganciclovir (GCV)). In this example, ALINK-modified host cells 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 S phase, which leads to the termination of DNA elongation and cellular apoptosis (Halloran and Fenton, 1998).

[0235] A modified HSV-TK gene (Preuss et al., 2010) is disclosed herein as an example of DNA encoding a negative selection marker that can be used in ALINK gene modification to selectively ablate cells with undesirable cell division rates.

[0236] It is contemplated herein that alternative and / or additional negative selection systems can be used with the tools and / or methods described herein. A variety of negative selection marker systems are known in the art (e.g., dCK.DM (Neschadim et al., 2012)).

[0237] For example, various clinically relevant negative selection systems are under development in the field of "gene-directed enzyme / prodrug therapy" (GEPT), which aims to improve the therapeutic efficacy of conventional cancer therapies with minimal or no side effects (Hedley et al., 2007; Nawa et al., 2008). In many cases, GEPT involves the use of viral vectors to deliver genes that are not present in mammalian cells to cancer cells or to the vicinity of cancer cells within the cancer cell region, producing an enzyme that can convert a relatively non-toxic prodrug into a toxic substance.

[0238] HSV-TK / GCV, cytosine deaminase / 5-fluorocytosine (CD / 5-FC), and carboxylesterase / irinotecan (CE / CPT-11) are examples of negative selection marker systems that have been evaluated in GEPT preclinical and clinical trials (Danks et al., 2007; Shah, 2012).

[0239] To address 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 the human deoxycytidine kinase enzyme to be thymidine-active and act as a negative selection (suicide) system with the non-toxic prodrugs bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), or L-deoxyuridine (LdU) (Neschadim et al., 2012).

[0240] The CD / 5-FC negative selectable 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., 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 protozoa and fungi, but is absent in mammalian cells. 5-Fluorocytosine (5-FC) is an antifungal prodrug that causes low levels of cytotoxicity in humans (Denny, 2003). CD catalyzes the conversion of 5-FC to the genotoxic substance 5-FU, which has high levels of toxicity in humans (Ireton et al., 2002).

[0241] The CE / CPT-11 system is based on the enzyme carboxylesterase, a serine esterase found in various tissues of mammalian species (Humerickhouse et al., 2000). The anticancer drug CPT-11 is activated by CE to produce an active compound called 7-ethyl-10-hydroxycamptothecin (SN-38), a potent mammalian topoisomerase I inhibitor (Wierdl et al., 2001). SN-38 induces the accumulation of double-stranded DNA breaks in dividing cells (Kojima et al., 1998).

[0242] Another example of a negative selection marker system is the iCasp9 / AP1903 suicide system, which uses modified human caspase-9 fused to human FK506-binding protein (FKBP) and allows chemical dimerization using the small molecule AP1903. This system has been safely tested in humans. Administration of the dimerization agent induces apoptosis in cells expressing the engineered caspase-9 component. This system has several advantages, including low potential immunogenicity; because it is composed of human gene products, the dimerization agent acts only on cells expressing the engineered caspase-9 component (Straathof et al., 2005). The iCasp / AP1903 suicide system is currently being tested in clinical settings (Di Stasi et al., 2011).

[0243] It is contemplated herein that the negative selection marker system of the ALINK system can be replaced with a growth antagonist system. As used herein, the term "growth antagonist" refers to a natural or engineered compound whose presence inhibits (fully or partially) cell division. For example, Omomyc ER is a fusion protein of a mutant mouse estrogen receptor (ER) domain and a MYC dominant-negative Omomyc. When tamoxifen (TAM) is induced, the fusion protein Omomyc EROmomyc is localized to the nucleus, where it dimerizes with C-Myc, L-Myc, and N-Myc, sequestering them in a complex that cannot bind to the Myc DNA-binding consensus sequence (Soucek et al., 2002). Cells lacking Myc activity are unable to divide (Oricchio et al., 2014). Another example of a proliferation antagonist is A-Fos, a dominant-negative heterodimer of the oncogenes Fos and Jun that inhibits DNA binding in equimolar competition with activator protein 1 (AP1) (Olive et al., 1997). A-Fos also fuses to the ER domain, resulting in its nuclear localization induced by TAMs. Omomyc ER / Tamoxifen or A-Fos ER / Tamoxifen can be used in place of TK / GCV to make ALINK.

[0244] Cells can also be modified to be "fail-safe" by operably linking a CDL to an EARC (e.g., an inducible activator-based gene expression system). Under these conditions, the CDL is expressed only in the presence of an inducer of the inducible activator-based gene expression system, and only in its presence can the cells divide. Under these conditions, EARC-modified cells cease division, significantly slow down division, or die in the absence of the inducer, depending on the mechanism of action of the inducible activator-based gene expression system and CDL function. Cells can be modified to contain homozygous or compound heterozygous EARC, or engineered so that only EARC-modified alleles are capable of producing functional CDL. In one embodiment, for example, EARC modifications can be introduced into all alleles of CDL to provide a mechanism for cell division control.

[0245] The EARC can be inserted into the CDL at any position that allows for the simultaneous expression of the CDL and the activator component of the inducible system in the presence of an inducer.

[0246] In one embodiment, an "activator"-based gene expression system is preferable to a "repressor"-based gene expression system. For example, when repressing CDL using a repressor, loss of function mutation of the repressor relieves CDL expression, thereby allowing cell proliferation. In the case of cell division activation-based repression, loss of activator function (mutation) stops CDL expression, thereby disabling cell proliferation.

[0247] In some embodiments, the EARC system is a dox-crosslinking system, a cumate switch induction system, an ecdysone induction system, an electromagnetic wave induction system, or a ligand reversible dimerization system.

[0248] A dox-bridge can be inserted into a CDL (e.g., CDK1) in a host cell such 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-bridged EARC can contain a reverse tetracycline transactivator (rtTA) gene under the transcriptional control of a promoter active in dividing cells (e.g., in CDL) (Urlinger et al., 2000). This targeted insertion results in the CDL promoter no longer being available for CDL transcription. To restore CDL transcription, a tetracycline response element promoter (e.g., a TRE (Agha-Mohammadi et al., 2004)) can be inserted in front of the CDL transcript, which will express the CDL gene only in the presence of rtTA and doxycycline. When the only source of CDL expression is the dox-bridged allele, CDL gene expression does not occur in the absence of doxycycline. Without CDL expression, EARC-modified cells are impaired in their proliferation either by cell death, cessation of cell division, or by slowing the rate of cell mitosis so that EARC-modified cells cannot contribute to tumor formation.

[0249] As used herein, the term "dox-bridge" refers to a mechanism for separating promoter activity from a target transcription region by expressing rtTA from an endogenous or exogenous promoter (Gossen et al., 1995) and placing transcription of the target region under the control of a TRE. As used herein, "rtTA" refers to the reverse tetracycline transactivator element of the tetracycline-inducible system (Gossen et al., 1995), and "TRE" refers to a promoter composed of a TetO operator sequence upstream of a minimal promoter. After rtTA binds to the TRE promoter in the presence of doxycycline, transcription of the locus downstream of the TRE promoter increases. The rtTA sequence can be inserted in the same transcription unit as the CDL or at a different location in the genome, as long as the permissive or non-permissive status of transcriptional expression of the target region is controlled by doxycycline. Dox-bridge is an example of an EARC.

[0250] The introduction of the EARC system into the 5' regulatory region of a CDL is also contemplated herein.

[0251] It is contemplated herein that alternative and / or additional inducible activator-based gene expression systems can be used with the tools or methods described herein to generate EARC modifications.

[0252] For example, when cell division and / or proliferation is desired, a destabilizing protein domain (Banaszynski et al., 2006) fused to the functional protein product of the encoded CDL can be used together with a small molecule synthetic ligand to stabilize the CDL fusion protein. In the absence of a stabilizing substance, the destabilized CDL protein would be degraded by the cell, which would then cease proliferation. When a stabilizing compound is added, it binds to the destabilized CDL protein, preventing it from being degraded, allowing the cell to proliferate.

[0253] For example, transcription activator-like effector (TALE) technology (Maeder et al., 2013) can be combined with dimerization-regulated expression induction (Pollock and Clackson, 2002). Using TALE technology, a DNA-binding domain designed to be specific for a sequence can be created that is placed alongside a minimal promoter that replaces the CDL promoter. The TALE DNA-binding domain was also extended with a drug dimerization domain. The latter can then be bound to another engineered protein with the corresponding dimerization and transcription activation domains.

[0254] For example, a reverse cumate transactivator (rcTA) can be inserted into the 5' untranslated region of CDL, allowing it to be expressed by the endogenous CDL promoter. Six repeats of the cumate operator (6xCuO) can be inserted immediately before the transcription start (ATG) of CDL. In the absence of cumate in the system, rcTA cannot bind to 6xCuO, and CDL is not transcribed because 6xCuO is inactive. When cumate is added, it forms a complex with rcTA, allowing it to bind to 6xCuO, thereby enabling CDL transcription (Mullick et al., 2006).

[0255] For example, retinoid X receptor (RXR) and an N-terminally truncated version of ecdysone receptor (EcR) (VpEcR) fused to the activation domain of Vp16 can be inserted into the 5' untranslated region of CDL, allowing them to be coexpressed by the endogenous CDL promoter. An ecdysone response element (EcRE) can be inserted into CDL directly upstream of the start codon, along with a downstream minimal promoter. Coexpressed 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 the EcRE, and CDL is not transcribed. In the presence of ecdysone or muristerone A, the dimerized RXR / VpEcR binds to the EcRE, allowing CDL to be transcribed (No et al., 1996).

[0256] For example, transient receptor potential vanilloid-1 (TRPV1) can be inserted into the 5' untranslated region of CDL together with ferritin and co-expressed by the endogenous CDL promoter. Alternatively, a promoter inducible by NFAT (NFATre) can be inserted into CDL directly upstream of the start codon. Under normal circumstances, the NFAT promoter is inactive. However, upon exposure to low-frequency radio waves, TRPV1 and ferritin can induce Ca ions entering the cell. ++ This then converts cytoplasmic-NFAT (NFATc) to nuclear-NFAT (NFATn), which ultimately activates NFATre to transcribe CDL ( Stanley et al., 2015 ).

[0257] For example, the CDL can be functionally divided into portions / domains: a 5'-CDL and a 3'-CDL, and an FKBP peptide sequence can be inserted into each domain. An IRES (internal ribosome entry site) sequence can be placed between the two domains, which are simultaneously transcribed by the CDL promoter but produce two separate proteins. In the absence of an inducer, the two separate CDL domains are functionally inactive. Upon introduction of a dimerizing agent such as rapamycin or AP20187, the FKBP peptide dimerizes, resulting in the 5'- and 3'-CDL portions and reconstituting an active protein (Rollins et al., 2000).

[0258] In an embodiment of this method, the genetically modified cell comprises a set of transgenes, each transgene encoding a cytoplasmic, membrane-associated, or locally acting gene product whose function is to reduce the function of graft-attacking leukocytes and NK cell activation, or to act as a defense mechanism against leukocyte attack.

[0259] Methods for genetically modifying cells to contain at least one mechanism for providing local immunosuppression at the transplant site when the cells or a population of such cells are transplanted into an allogeneic host are described, for example, in WO 2016 / 141480, the entire teachings of which are incorporated herein by reference.

[0260] The set 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 an embodiment, the set of transgene genes includes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0261] Optionally, the method further includes expressing one or more of the following transgenes in the cells: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39. In embodiments, the TGF-β or biological agent acts locally within the graft environment.

[0262] Techniques for introducing various genetic modifications, such as transgenes, into animal cells are described herein and are generally known in the art.

[0263] In embodiments of this method, the cell is a stem cell, a cell amenable to genome editing, or a cell that can serve as a source of therapeutic cell types (e.g., a cell that can be directed to differentiate into lineage-restricted or terminally differentiated cells that can be used in cell therapy, or a cell of a desired target tissue). In embodiments, 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 breast stem cell, an olfactory adult stem cell, a hair follicle stem cell, a multipotent stem cell, an amniotic 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, e.g., a mammalian cell, such as a human or mouse cell.

[0264] Techniques for transplanting genetically modified cells into the transplant site of an allogeneic host are described herein and are known in the art.

[0265] In various embodiments of any of the disclosed methods, the host has a degenerative disease or condition that can be treated using cell therapy. Examples of such diseases or conditions include, in various embodiments, 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, neurological diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS, enzyme or hormone deficiency, metabolic disorder (e.g., lysosomal storage disorder, 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, Sjogren's syndrome, Hashimoto's thyroiditis, vasculitis, autoimmune diseases, and the like). immune 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, 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, keratoglobuloid, muscular dystrophy, neuronal ceroid lipofuscinosis, previous disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age-related diseases (e.g., atherosclerosis, cardiovascular disease (e.g., angina pectoris, myocardial infarction), cataracts, osteoporosis, or hypertension).

[0266] Pharmaceutical Composition The cloak cells described herein can be incorporated into a vehicle for administration to a patient, such as a human patient undergoing a transplant or suffering from a disease or condition described herein. Pharmaceutical compositions containing cloak cells can be prepared using methods known in the art. For example, such compositions can be prepared in a desired form (e.g., in the form of an aqueous solution) using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013); incorporated herein by reference).

[0267] The cloak cells described herein can be administered in any physiologically compatible carrier (e.g., a buffered saline solution). Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art. Other examples include liquid 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, as well as in oils. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. This solution is preferably sterile and fluid to the extent that easy injectability 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 through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosol, etc. The solution of the present invention can be prepared by using a pharmaceutically acceptable carrier or diluent and, if necessary, other ingredients listed above, followed by filtration sterilization, and then incorporating the cloak cells described herein.

[0268] For example, solutions containing the pharmaceutical compositions described herein may be suitably buffered if necessary, and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. In any event, the person responsible for administration will determine the appropriate dose for each individual subject. Furthermore, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Sciences standards.

[0269] Pharmaceutical compositions comprising cloak cells in a semi-solid or solid carrier are typically formulated for surgical implantation at a transplant site or at the site of a disease or condition in a subject. It is understood that liquid compositions may also be administered via a surgical procedure. In certain embodiments, semi-solid or solid pharmaceutical compositions may comprise semi-permeable gels, matrices, cell scaffolds, etc., which may be non-biodegradable or biodegradable. For example, in certain embodiments, it may be desirable or appropriate to isolate cloak cells from their surroundings while still allowing the cells to secrete and deliver biological molecules (e.g., therapeutic agents listed in Table 2) to surrounding cells.

[0270] In other embodiments, different types of degradable gels and networks are utilized for 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.

[0271] In another embodiment, one or more hydrogels are used in the pharmaceutical composition. The one or more hydrogels may include collagen, atelocollagen, fibrin constructs, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, and poly(ethylene oxide). Additionally, hydrogels may be formed from poly(2-hydroxyethyl methacrylate), poly(acrylic acid), self-assembling peptides (e.g., RAD16), poly(methacrylic acid), poly(N-vinyl-2-pyrrolidinone), poly(vinyl alcohol), and their copolymers with each other and with hydrophobic monomers such as methyl methacrylate and vinyl acetate. Hydrophilic polyurethanes containing large poly(ethylene oxide) blocks are also preferred. Other preferred materials include hydrogels comprising interpenetrating networks of polymers, which may be formed by addition or condensation polymerization, and whose components may include hydrophilic and hydrophobic monomers such as those listed above. In situ-forming degradable networks are also suitable for use in the present invention (see, e.g., Anseth, K.S. et al., J. Controlled Release, 2002; 78:199-209; Wang, D. et al., Biomaterials, 2003; 24:3969-3980; U.S. Patent Application Publication No. 2002 / 0022676). These in situ-forming materials are formulated as fluids suitable for injection and can then be induced to form hydrogels by various means, such as changes in temperature, pH, and exposure to light in situ or in vivo. In one embodiment, the construct contains a fibrin glue-containing gel. In another embodiment, the construct contains an atelocollagen-containing gel.

[0272] The polymer used to form the matrix can be in the form of a hydrogel. Generally, hydrogels are crosslinked polymeric materials that can absorb more than 20% of their weight in water while maintaining a well-defined three-dimensional structure. This definition includes dry crosslinked polymers and water-swollen materials that swell in an aqueous environment. A host of hydrophilic polymers can be crosslinked to produce hydrogels, whether the polymers are biologically derived, semi-synthetic, or fully synthetic. Hydrogels can be manufactured from synthetic polymeric materials. Such synthetic polymers can be tailored to a range of properties and predictable lot-to-lot uniformity, and generally represent a reliable source of materials without immunogenicity concerns. The matrix can include hydrogels formed from self-assembling peptides (e.g., those discussed in U.S. Pat. Nos. 5,670,483 and 5,955,343, U.S. Patent Application No. 2002 / 0160471, and PCT Publication WO 02 / 062969).

[0273] Properties that make hydrogels valuable in drug delivery applications include equilibrium swelling, sorption kinetics, solute permeability, and their in vivo performance characteristics. Permeability to compounds depends in part on swelling or water content and biodegradation rate. Because the mechanical strength of gels can decrease proportionally with swelling, it is well within the contemplation of the present invention that hydrogels can be attached to substrates to enhance the mechanical strength of composite systems. In some embodiments, hydrogels can be impregnated into porous substrates to obtain the mechanical strength of the substrate along with the useful delivery properties of the hydrogel.

[0274] In another embodiment, the pharmaceutical composition comprises a biocompatible matrix made from natural, modified natural, or synthetic biodegradable polymers, including homopolymers, copolymers, and block polymers, and combinations thereof.

[0275] Examples of suitable biodegradable polymers or polymer classes include any biodegradable polymer discussed within this disclosure, including, but not limited to, fibrin, types I, II, III, IV, and V collagen, elastin, gelatin, vitronectin, fibronectin, laminin, thrombin, poly(amino acids), oxidized cellulose, tropoelastin, silk, ribonucleic acid, deoxyribonucleic acid; proteins, polynucleotides, gum arabic, reconstituted basement membrane matrix, starch, dextran, alginate, hyaluron, chitin, chitosan, agarose, polysaccharides, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, derivatives and mixtures thereof. Suitable polymers also include poly(lactide) (PLA), which can be formed from L(+) and D(-) polymers, polyhydroxybutyrate, polyurethane, polyphosphazene, poly(ethylene glycol)-poly(lactide-co-glycolide) copolymer, degradable polycyanoacrylate, and degradable polyurethane. 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.

[0276] 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 esters, polyoxaesters containing amine groups, poly(propylene fumarate), polyfumarates, polydioxanone, 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(para-carboxyphenoxy)propane and sebacic acid, sebacic acid copolymers, copolymers of caprolactone, poly(lactic acid) / poly(glycolic acid) / polyethylene glycol copolymers, copolymers of polyurethane and poly(lactic acid), copolymers of α-amino acids, copolymers of α-amino acids and caproic acid, copolymers of α-benzyl glutamate and polyethylene glycol, copolymers of succinate and poly(glycol), polyphosphazenes, poly(hydroxyalkanoates), and mixtures thereof. Binary and ternary systems are also contemplated.

[0277] In general, the material used to form the matrix is ​​desirably configured to (1) have mechanical properties suitable for the intended use, (2) remain sufficiently intact for tissue ingrowth and healing, (3) not cause an inflammatory or toxic response, (4) be metabolized in the body after achieving its purpose, (5) be easily processed into the desired end product to be formed, (6) exhibit an acceptable shelf life, and (7) be easily sterilized.

[0278] In another embodiment, the population of cloak cells can be administered using a scaffold. The scaffold can have any of the compositions, shapes, and porosities described above. Typically, these three-dimensional biomaterials contain living cells embedded in an extracellular matrix attached to, dispersed within, or entrapped in the scaffold. When implanted in a target area of ​​the body, these implants integrate with the host tissue, and the implanted cells gradually become established.

[0279] Non-limiting examples of scaffolds that can be used include textile structures such as woven fabrics, knitted fabrics, braided fabrics, meshes, nonwoven fabrics, and warp knitted fabrics; porous foams, semi-porous foams, perforated films or sheets, microparticles, beads, and spheres, as well as composite structures that are combinations of the above structures. Nonwoven mats can be formed using fibers made of synthetic absorbable copolymers of glycolic acid and lactic acid (PGA / PLA), such as those sold under the trade name VICRYL suture (Ethicon, Inc., Somerville, NJ). Foams made of poly(ε-caprolactone) / poly(glycolic acid) (PCL / PGA) copolymers formed by processes such as freeze-drying or lyophilization, as discussed in U.S. Pat. No. 6,355,699, can also be utilized.

[0280] In another embodiment, the framework is a felt, which can be composed of multifilament yarns made from bioabsorbable materials. The yarns can be felted using standard textile processing techniques consisting of crimping, cutting, carding, and needling. In another embodiment, cells are seeded onto a foam scaffold, which can be used as a composite structure.

[0281] The framework can be molded into a shape useful for filling a tissue void, for example. Thus, the framework can be shaped to provide channels for nerve growth as well as a scaffold for supporting and surrounding tissue, such as vascular tissue, muscle tissue, etc. Furthermore, it will be appreciated that populations of cells can be cultured on preformed, non-degradable surgical or implantable devices.

[0282] Pharmaceutical compositions may include preparations made from cloak cells formulated with a pharmaceutically acceptable carrier or vehicle. Suitable pharmaceutically acceptable carriers include any of those discussed within this disclosure, including, but not limited to, water, salt solutions (such as Ringer's solution), alcohol, oil, gelatin, polyvinylpyrrolidine, carbohydrates such as lactose, amylose, or starch, fatty acid esters, and hydroxymethylcellulose. Such preparations may be sterilized and, if necessary, mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring agents). Pharmaceutical carriers suitable for use in the present invention are known in the art and are described, for example, in Pharmaceutical Sciences (17 th Ed., Mack Pub. Co., Easton, Pa.) and WO 96 / 05309.

[0283] Treatment method The cloak cells and compositions described herein can be administered to a subject in need thereof (e.g., a subject receiving or who has received a transplant or a subject with a disease or condition described herein) by a variety of routes, including local administration at or near the site of transplant, local administration to the site affected by the disease or condition (e.g., injection into a joint to treat RA, injection into a joint to treat wet AMD, direct administration to the central nervous system (CNS) (e.g., intracerebral, intraventricular, intrathecal, intracisternal, or stereotactic administration to treat neurological disorders such as Parkinson's disease), direct injection into the myocardium to treat myocardial infarction, intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most appropriate route for administration in any given case will depend on the particular cells or composition being administered, the patient, the pharmaceutical formulation, the method of administration (e.g., time and route of administration), the patient's age, weight, and sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. The composition may 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, cloak cells may be administered by any means that places the cell population at the desired location, including by implantation using a catheter, syringe, shunt, stent, microcatheter, pump, device, or scaffold.

[0284] As described herein, prior to administration, the cell population can be incubated in the presence or under conditions of one or more factors that stimulate stem cell differentiation into a desired cell type (e.g., neurons, cardiomyocytes, RPE cells, insulin-producing cells, blood clotting factor-producing cells, articular fibroblasts, or other cell types described herein). Such factors are known to those skilled in the art, and those skilled in the art will understand that determining appropriate conditions for differentiation can be accomplished using routine experimentation. Such factors include growth or trophic factors, chemokines, cytokines, cell products, demethylating agents, and other stimuli known to stimulate stem cell differentiation along, for example, angiogenic, hemangiogenic, vasculogenic, skeletal muscle, vascular smooth muscle, pericyte, neuronal, or vascular endothelial pathways or lineages. Alternatively, the composition administered to the patient comprises a population of cloak cells with one or more factors that stimulate cell differentiation into a desired cell type, where cell differentiation occurs in vivo at the tissue site. In some embodiments, cloak cells can be differentiated into an organ or tissue in vitro using methods known to those skilled in the art and administered to a subject in need of an organ or tissue transplant.

[0285] In some embodiments, cells of a particular cell type are collected from a patient or donor (e.g., from an HLA-matched or mismatched donor, e.g., free of a disease or condition), modified to express one or more (e.g., one, two, three, four, five, six, seven, or eight) cloaking transgenes, and then administered to a subject. Such an approach is useful for treating subjects carrying mutations in particular genes or for subjects deficient in particular secreted proteins or enzymes (e.g., using cloaked donor cells that endogenously express the protein or enzyme that is deficient in the subject), because the cloaked donor cells can endogenously express the wild-type version of the gene. This approach can also be used to treat subjects undergoing organ or tissue transplants, because 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) cloaking transgenes before the transplant is performed.

[0286] Subjects who can be treated as described herein include those who have received a transplant or have a disease or condition described herein (e.g., wet AMD or retinal dystrophy, neurodegenerative disease (e.g., Parkinson's disease), myocardial infarction, osteoarthritis or RA, diabetes, hemophilia, metabolic disorders, or a disease or condition listed in Table 2). The cells, compositions, and methods described herein can be used to treat diseases or conditions caused by or associated with cell loss, protein mutation or deficiency, or abnormal protein production, which can be treated using cell replacement proteins or cell therapy, production of therapeutic proteins, production of agonistic antibodies, or production of inhibitory antibodies. The methods described herein can include screening the subject for mutations in genes associated with defective protein production before treatment or administration with a composition described herein. Subjects can be screened for genetic mutations using standard methods known to those of skill in the art (e.g., genetic testing). The methods described herein can also include evaluating the symptoms of the disease or condition in the subject before treatment or administration with a cloak cell or composition described herein. The subject can then be evaluated using the same diagnostic test after administration of the cloak cells or composition to determine whether the subject's condition has improved. The compositions and methods described herein can be administered as a prophylactic treatment to patients who have undergone tissue or organ transplantation before the patient shows signs of tissue or organ rejection.

[0287] The cloak cells, compositions, and methods described herein can be used to replace dead or dying cells in a subject (e.g., to replace neurons in a subject suffering from a neurodegenerative disease or to replace cardiomyocytes in a subject who has had a myocardial infarction). The cloak cells, compositions, and methods described herein can also be used to provide immunosuppression in the area of ​​tissue or organ transplantation or to reduce the risk of tissue or organ transplant rejection. Cloak 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 supply wild-type versions of proteins that are mutated or missing in a subject (e.g., proteins that are produced but do not function properly due to a genetic mutation, such as truncated proteins or proteins with altered charge, polarity, or binding properties; or proteins that are not produced or are produced in insufficient amounts, such as defective protein production associated with a disease or condition in Table 2). Cloak cells expressing a therapeutic agent (e.g., an inhibitory or neutralizing antibody), compositions comprising such cells, or methods of administering such cells can be used to block or neutralize an overexpressed or abnormally produced protein in a subject (e.g., a protein produced at a time or location different from the production of that protein in a healthy subject (e.g., aberrant protein production associated with a disease or condition listed in Table 2)).

[0288] Treatment may include the administration of various unit doses of cloak cells or compositions containing cloak cells. Each unit dose typically contains a predetermined amount of cloak cells as described herein. The amount administered and the specific route and formulation of administration are within the skill of those in the clinical arts. The unit dose need not be administered as a single infusion, but may include continuous infusion over a set period of time. Administration may be performed using a catheter, syringe, shunt, stent, microcatheter, pump, implantation using a device, or implantation using a scaffold. The number of cells administered may vary depending on whether the cells are administered to a tissue, organ, or body site associated with disease or injury, or subcutaneously to produce cloaked subcutaneous tissue. For administration to a tissue, organ, or body site, cloak cells may be administered in a concentration of, for example, 1 x 10 4 cells ~ 1 x 10 10 cells (e.g., 1 x 10 4 , 2 × 10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2 × 10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×10 5 , 9×10 5 , 1×10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7, 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 2 × 10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 The number of cells administered depends on the size of the recipient tissue, organ, or body part. For example, 2.5 x 10 4 ~1×10 5 cells (e.g., 2.5 x 10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 or 1 x 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 ~1×10 8 cells (e.g., 1 x 10 6 , 2 × 10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 1×10 7 , 2 × 10 7 , 3×10 7 , 4×10 7 , 5×10 7, 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 or 1 x 10 8 5 x 10 cells) can be administered (e.g., injected) into the joint, the amount of cells depends on the size of the joint; and 5 x 10 8 ~5×10 9 cells (e.g., 5 x 10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 or 5 x 10 9 To create a cloaked subcutaneous tissue, 8 × 10 cells can be administered into the myocardium. 8 cells ~3×10 9 cells (e.g., 8 x 10 8 , 9×10 8 , 1×10 9 , 2 × 10 9 , 3×10 9 The cloak cells can be administered subcutaneously (e.g., injected). The cloak cells can be administered in two or more doses (e.g., 2, 3, 4, 5, or more different doses, e.g., into different sized joints in a patient with RA) or the same dose two or more times (e.g., 2, 3, 4, 5, 6, or more times over the course of an hour, day, week, month, or year). In some embodiments, the cloak cells described herein are administered as tissue (e.g., tissue expanded and / or differentiated in vitro from cloak cells). In some embodiments, the cloak tissue is administered (e.g., implanted) with a gel, biocompatible matrix, or scaffold.

[0289] The compositions described herein can be used to prevent or reduce transplant rejection or to ameliorate the symptoms of a disease or condition listed in Table 2 (e.g., to reduce the symptoms of osteoarthritis or RA (e.g., to reduce inflammation, joint pain, stiffness, joint pain, stiffness, or immobility); to reduce the symptoms of retinal dystrophy or wet AMD (e.g., to improve vision, slow or reduce ocular neovascularization); to reduce the symptoms of Parkinson's disease (e.g., to reduce tremor, stiffness, bradykinesia, or to improve posture or gait); to reduce the symptoms of diabetes (e.g., to improve insulin resistance, to reduce insulin resistance, or to improve insulin resistance)). The cloak cells or compositions described herein may be administered in an amount sufficient to improve phospholipid levels, reduce the need for regular insulin injections; reduce symptoms of myocardial infarction (e.g., improve cardiac function, reduce infarct size); reduce symptoms of hemophilia (e.g., increase levels of blood clotting factors such as Factor VIII, reduce excessive bleeding, reduce bruising, reduce nosebleeds, reduce joint pain or swelling); or reduce symptoms of metabolic disorders (e.g., increase appetite, growth, or weight gain, or reduce lethargy, weight loss, jaundice, seizures, abdominal pain, or vomiting). Transplant rejection can be assessed 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 cloak cells or compositions described herein results in transplant rejection outcomes comparable to those observed in subjects receiving immunosuppressants. Symptoms of the diseases and conditions described herein can be assessed using standard methods known to those of skill in the art and can be reduced (e.g., the subject's condition can improve) 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 cloak 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 more after administration of the compositions described herein.Depending on the dose and route of administration used for treatment, patients may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more after administration of the cloak cells or composition. Depending on the results of the evaluation, patients may receive additional treatment.

[0290] Combination therapy In some embodiments, the cloak cells described herein are administered in combination with one or more additional therapeutic agents. The additional therapeutic agent can be administered before, after, or simultaneously with the administration of the cloak cells. The cloak cells and the additional therapeutic agent can also be administered simultaneously via a co-formulation. The cloak cells and the therapeutic agent can also be administered sequentially, such that the actions of the cloak cells and the therapeutic agent overlap and their combined effect results in a reduction in symptoms or other parameters associated with the disorder that is greater than that observed with the cloak cells or the therapeutic agent delivered alone or greater than that observed in the absence of the other. The effects of the cloak cells and the therapeutic agent can be partially additive, fully additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of the cloak cells and the therapeutic agent can be achieved by any appropriate route, including, but not limited to, oral, intravenous, intramuscular, topical, or subcutaneous. The cloak cells and the therapeutic agent can be administered by the same or different routes. For example, cloak cells can be administered by subcutaneous injection while the additional therapeutic agent is administered orally. The cloak cells can be administered immediately before or immediately after the additional therapeutic agent, 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, up to 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 1 to 7, 1 to 14, 1 to 21, or 1 to 30 days before or after the additional therapeutic agent.

[0291] In one example, the additional therapeutic agent is an immunosuppressant commonly given in organ or tissue transplants. The immunosuppressant can be an agent given immediately after transplant to prevent acute rejection (e.g., methylprednisolone, Atgam, thymoglobulin, OKT3, basiliximab, or daclizumab) or an immunosuppressant used for maintenance (e.g., prednisone, calcineurin inhibitors (e.g., cyclosporine, tacrolimus), mycophenolate mofetil, azathioprine, or rapamycin). Other immunosuppressive agents given after organ transplantation include corticosteroids (e.g., methylprednisolone, dexamethasone, prednisolone), cytotoxic immunosuppressants (e.g., azathioprine, chlorambucil, cyclophosphamide, mercaptopurine, methotrexate), immunosuppressive 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, cloak cells are administered at or near the transplant site, or the transplanted tissue is modified to express one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cloaking transgenes, and immunosuppressive agents are administered as an additional source of immunosuppression, if necessary.

[0292] For use in treating inflammatory and autoimmune-related diseases or conditions, the additional agent can be a disease-modifying antirheumatic drug (DMARD), a biological response modifier (a type of DMARD), a corticosteroid, or a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, the additional agent is a biologic such as prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, or tofacitinib, adalimumab, abatacept, anakinra, kineret, cetrolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab. In some embodiments, the additional agent is 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab (Lemtrada), aminosalicylic acid (5-aminoalicylic acid, sulfasalazine, mesalamine, balsalazide, olsalazine), antibiotic, antihistamine, anti-TNFα (infliximab, adalimumab, certolizumab pegol, natalizumab), azathioprine, belimumab, beta interferon, calcineurin inhibitor, cetrolizumab, corticosteroid, cromolyn, cyclosporine A, cyclosporine, dimethyl fumarate (Tecfidera), etanercept, fingolimod (Diflunisone), Renia), fumarate esters, glatiramer acetate (Copaxone), golimumab, hydroxyurea, IFNγ, IL-11, infliximab, leflunomide, leukotriene receptor antagonists, long-acting beta-2 agonists, mitoxantrone, mycophenolate mofetil, natalizumab (Tysabri), ocrelizumab, pimecrolimus, probiotics (VSL#3), retinoids, rituximab, salicylic acid, short-acting beta-2 agonists, sulfasalazine, tacrolimus, teriflunomide (Aubagio), theophylline, tocilizumab, ustekinumab (anti-IL-12 / IL-23), or vedolizumab (anti-α3β7 integrin). In this case, cloak cells may be administered to replace tissues or organs damaged by inflammatory or autoimmune-related diseases or conditions.In another example, the administered cloak cells may be modified to express a biological therapeutic agent (e.g., an antibody) directed to the treatment of a particular inflammatory or autoimmune-related disease or condition, and the additional agent may be a chemical compound or a general anti-inflammatory agent (e.g., an NSAID or corticosteroid).

[0293] 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, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab. The administered cloak cells may be cartilage- or bone-producing cells of the joint. In some embodiments, the cloak cells may be modified to produce anti-TNFα antibodies and may be administered in combination with an anti-inflammatory agent (e.g., a corticosteroid).

[0294] In another example, for use in treating AMD or retinal dystrophy, the additional therapeutic agent can be an additional biological agent (e.g., bevacuzimab, ranibizumab, or aflibercept), photodynamic therapy, or photocoagulation. The administered cloak cells can be retinal cells (e.g., RPE cells). In some embodiments, the cloak cells can be modified to produce a VEGF inhibitor and can be administered in combination with photodynamic therapy or photocoagulation.

[0295] For use in treating Parkinson's disease, the cloak cells described herein can be administered with carbidopa-levodopa, dopamine agonists (e.g., pramipexole, ropinirole, rotigotine, or apomorphine), MAO-B inhibitors (e.g., selegiline or rasagiline), catechol-O-methyltransferase inhibitors (e.g., entacapone or tolcapone), anticholinergics (e.g., benztropine or trihexyphenidyl), amantadine, or deep brain stimulation. The administered cloak cells can be dopaminergic neurons.

[0296] Additional agents for treating 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, trandrapril, angiotensin II receptor blockers (e.g., candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan), angiotensin II receptor neprilysin inhibitors (e.g., sacubitril / valsartan), beta-blockers (e.g., acebuterol, atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, sotalol), combinations of 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), nicotinic acid (e.g., lovastatin), cholesterol absorption inhibitors (e.g., ezetimibe / simvastatin), digitalis preparations (e.g., lanoxin), diuretics (e.g., amiloride, bumentanide, chlorothiazide, chlorthalidone, furosemide, halothiazide, These include vasodilators (e.g., isosorbide dinitrate, nesiritide, hydralazine, nitrates, minoxidil), dual antiplatelet therapy (e.g., aspirin and a P2Y12 inhibitor), or cardiac procedures (e.g., angioplasty, prosthetic heart valve surgery, atherectomy, bypass surgery, cardiomyoplasty, heart transplant, minimally invasive cardiac surgery, radiofrequency ablation, stenting, or transmyocardial revascularization). The administered cloak cells may be cardiomyocytes.

[0297] For use in treating an infectious disease, 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 cloak cells can be immune cells (e.g., cells that can help combat an infection (e.g., cloak T cells or B cells)).

[0298] For use in the treatment of 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), an SGLT2 inhibitor (e.g., canagliflozin, dapagliflozin), an α-glucosidase inhibitor (e.g., acarbose, miglitol), a bile acid sequestrant (e.g., colesevelam), aspirin, or dietary therapy. The administered cloak cells may be pancreatic β cells, which may optionally be modified to express a transgene encoding insulin.

[0299] For use in the treatment of hemophilia, the additional therapeutic agent may be a clotting factor, desmopressin, a clot-preserving agent (e.g., an antifibrinolytic agent such as aprotinin, aminocaproic acid, fibrigogen, or tranexamic acid), a fibrin sealant, or physical therapy. The administered cloak cells may be liver sinusoidal cells or endothelial cells, which may optionally be modified to express a transgene encoding Factor VIII.

[0300] For the treatment of metabolic disorders or diseases, the additional therapeutic agent may be a coenzyme (e.g., biotin, hydroxycobalamin, riboflavin, pyridoxine, folic acid, thiamine, ubiquinone, tetrahydrobiopterin), bone marrow transplant, organ transplant (e.g., liver, kidney, or heart transplant), hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium-chain triacylglycerol, miglustat, enzyme replacement therapy, or dietary restriction (e.g., a low-protein or phenylalanine-restricted diet for a subject with phenylketonuria). Cloak cells may be cells that carry a wild-type copy of a gene that is mutated in a subject with a metabolic disorder or cells that endogenously produce an enzyme that is deficient in a subject with a metabolic disorder (e.g., liver cells, kidney cells, heart cells, or any other cells that carry a wild-type copy of a gene that is mutated in a subject with a metabolic disorder or produce an enzyme that is deficient in a subject with a metabolic disorder).

[0301] For use in treating cancer, the additional agent may be a checkpoint inhibitor, a chemotherapeutic agent, a biological drug, a non-drug therapy (e.g., radiation therapy, cryotherapy, hyperthermia, or surgical removal of tumor tissue), or an anti-cancer vaccine. Cloak cells may be immune cells (e.g., macrophages, natural killer cells, dendritic cells, or T cells) that can help combat cancer.

[0302] Checkpoint inhibitors can be classified into at least four major categories: i) antibody-like agents that block inhibitory pathways directly on T cells or natural killer (NK) cells (e.g., PD-1 targeting antibodies 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) antibody-like agents that activate stimulatory pathways directly on T cells or NK cells (e.g., antibodies targeting OX40, GITR, or 4-1BB); iii) antibody-like agents that block inhibitory pathways on immune cells or deplete inhibitory populations of immune cells (e.g., CTLA-4 targeting antibodies such as ipilimumab or tremelimumab, antibodies targeting VISTA, and PD-L2 (e.g., AMP-1 receptor agonists). 224), antibodies targeting Gr1 or Ly6G); iv) agents such as antibodies or small molecules that directly block inhibitory pathways on cancer cells or rely on antibody-dependent cellular cytotoxicity to enhance cytotoxicity against 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 checkpoint inhibitor is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of HVEM, CD160, CHK1, CHK2, a B-7 family ligand, or a combination thereof. 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 checkpoint inhibitor 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 another embodiment, the checkpoint inhibitor is a fusion protein, e.g., an Fc receptor fusion protein.In some embodiments, the checkpoint inhibitor is an agent, such as an antibody, that interacts with a checkpoint protein, hi other embodiments, the checkpoint inhibitor is an agent, such as an antibody, that interacts with a ligand of the checkpoint protein.

[0303] Chemotherapeutic agents include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopyyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione-substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, corticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. Also included are 5-fluorouracil (5-FU), leucovorin (LV), irenotecan, oxaliplatin, capecitabine, paclitaxel, and doxetaxel. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneamines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its analogs adzelesin, carzelesin, and bizen). cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine;For example, enediyne antibiotics (e.g., antibiotics such as calicheamicin, especially calicheamicin gamma and calicheamicin omegal; dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramicin, azaserine, bleomycin, cactinomycin, carabicin (c arabicin), caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; metabolic antagonists such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as donepterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as darabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyridinium analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone, propionate, epithiostanol, mepitiostane, and testolactone; adrenal antidote drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine;Demecolcine; Diaziquone; Elfomitin; Elliptinium acetate; Epothilone; Etoglucide; Gallium nitrate; Hydroxyurea; Lentinan; Lonidainine; Maytansinoids such as maytansine and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraeline; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; Razoxane; Rhizoxin; Schizofuran; Spirogermanium; Tenuazonic acid; Triazicone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); Urethane; Binde cin; dacarbazine; mannommustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., 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; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeroda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. Two or more chemotherapeutic agents can be used in a cocktail administered in combination with the cloak cells described herein. Suitable dosing regimens for combination chemotherapy are known in the art.

[0304] Anti-cancer biologics include cytokines used in cancer treatment (e.g., interferons or interleukins (e.g., IL-2)). In another embodiment, the biologic is an anti-angiogenic agent, e.g., an anti-VEGF agent, e.g., bevacizumab. In some embodiments, the biologic is an immunoglobulin-based biologic, e.g., a monoclonal antibody (e.g., a humanized antibody, fully human antibody, Fc fusion protein, or functional fragment thereof), that acts on a target to stimulate an anti-cancer response or antagonize an antigen important to the cancer. Such agents include rituximab; daclizumab; basiliximab; palivizumab; infliximab; trastuzumab; gemtuzumab ozogamicin; alemtuzumab; ibritumomab tiuxetan; adalimumab; omalizumab; tositumomab-I-131; efalizumab; cetuximab; bevacizumab; natalizumab; tosilicum; Antibody-drug conjugates include trastuzumab, panitumab, ranibizumab, eculizumab, certolizumab pegol, golimumab, canakinumab, ustekinumab, ofatumumab, denosumab, motavizumab, raxibacumab, belimumab, ipilimumab, brentuximab, vedotin, pertuzumab, ado-trastuzumab emtansine, and obinutuzumab. Antibody-drug conjugates are also included.

[0305] kit The invention also features kits containing cloak cells described herein (e.g., cloak cells expressing a set of cloaking transgenes described herein (e.g., one, two, three, four, five, six, seven, or eight 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 IFNγR1 d39. In some embodiments, the cloak cells are further modified to include one or more systems for regulating cell division (e.g., the ALINK or EARC systems) and / or therapeutic agents (e.g., transgenes encoding proteins or antibodies). The cloak cells may be provided in a pharmaceutical composition. The kit may further comprise a syringe for administration of the cloak cells or pharmaceutical composition and instructions for administering the cloak cells or pharmaceutical composition to treat a disease or condition described herein. [Example]

[0306] 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, and it is understood that by their exemplary nature, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0307] Example 1: Materials and Methods Construction of vectors expressing target genes essential for allo-resistance A plasmid containing the cDNA sequence of the gene involved in alloresistance was obtained as follows. PD-L1: Mount Sinai Hospital, clone #V102001 FasL: Mount Sinai Hospital, #75719 Cd47:Mount Sinai Hospital, #V75535 Cd200:GE Dharmacon, ID# 17470 H2-M3: Mount Sinai Hospital, Clone #8188 Ccl21: Mount Sinai Hospital, Clone #V77120 Mfge8: Mount Sinai Hospital, Clone #V72614 Spi6: Mount Sinai Hospital, Clone #V8907

[0308] Expression vectors containing these genes of interest (GOI) or luciferase enzymes were constructed using the Gateway cloning system (Thermo Fisher). Cd47, Ccl21, Mfge8, and Spi6 cDNAs were obtained containing attB sites flanking the cDNA. Primers were designed to amplify the cDNA sequences of H2-M3, Cd200, FasL, and PD-L1, adding attB sites (Figure 15(a)). After PCR amplification, the attB-containing cDNAs were recombined into the pDONR221 vector (Thermo Fisher, #1256017) by BP (recombination between attB and attP sites) to generate entry (pENTRY) clones (Figure 15(b)). The BP reaction involved mixing the attB-flanked transgene cDNA with the pDONR221 plasmid in a 1 mL tube together with buffer and the BP enzyme provided by Invitrogen, where the BP enzyme recombines the GOI into the docking site of the pDONR221 plasmid. Insertion of the transgene into the pDONR221 plasmid was confirmed by DNA sequencing (TCAG Sequencing Facility at the Centre for Applied Genomics, Toronto). The pENTRY clone containing the GOI was then recombined into the destination vector via a Gateway LR (recombination between the attL and attR sites) reaction ( Figure 15(c) ). The LR reaction involved mixing the GOI-containing pDONR221 plasmid with the destination vector in a 1 mL tube together with buffer and the LR enzyme provided by Invitrogen, where the LR enzyme recombines the GOI cassette from pDONR221 into the docking site of the destination plasmid. The destination vector used for all transgene constructions contains a CAGG promoter followed by a Gateway entry site, an internal ribosome entry site (IRES), and either 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 (Figure 15(d)) was confirmed by restriction enzyme digestion.

[0309] ES cell culture, transfection, selection and cloning 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 at 37°C and 5% CO2 in a standard cell culture incubator.

[0310] Transfection was performed using JetPRIME reagent (Polyplus, catalog #14-07) according to the manufacturer's protocol and was carried out in three steps: 1) transfection with the PD-L1-IRES-GFP destination vector alone, 2) transfection with all other transgenes carrying a puromycin selection marker, and 3) transfection with the eLuciferase-IRES-GFP transgene.

[0311] Step 1: Following transfection with PD-L1-IRES-GFP, cells were plated at low density, allowing for multiple rounds of proliferation after 5-6 days. Individual cell clones present as cell aggregates (colonies) were selected based on the intensity of GFP expression and then expanded as clonal cell cultures. Clones with the highest and most consistent GFP expression were selected for the next step.

[0312] Step 2: 24 hours after transfection with a transgene containing a puromycin selection marker, puromycin was added to the culture medium. On day 3, cells were plated at clonal density, and puromycin selection continued until individual colonies were picked and clonally expanded. A number of these clones were screened in vivo, and one capable of forming teratomas in an allogeneic setting was designated "NT2."

[0313] Step 3: NT2 cells were 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.

[0314] Assessment of transgene expression levels RNA was isolated from cultures grown on 30 mm culture 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 #Bio-98020 from Bioline, gene-specific primers, and RNA at a 1:50 dilution. Samples were plated into 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 CFX Manager 3.1 software and expression levels were calculated using Microsoft Excel.

[0315] Teratoma assay Matrigel Matrix High Concentration (Corning cat# 354248) was diluted 1:1 with cold DMEM medium on ice. 7 Cells were suspended in 500 μL of DMEM and an equal volume of Matrigel. 100 μL of the suspension (5 × 10 cells) was injected subcutaneously into each dorsal flank of B6N (syngeneic) or FVB / N (allogeneic) mice. The resulting teratomas formed 2–4 weeks after injection. The size of the teratomas was measured using calipers, and the volume was calculated using the formula V = (L × W × H) / 2. Tumors grew to approximately 500 mm. 2 The teratomas from the previously described experiments were grown to 500 mm , which is sufficiently resistant and large enough for downstream experiments. All of the transgenes were 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 for complete inhibition of cell division with administration of ganciclovir (GCV). Teratomas from previously described experiments grew to 500 mm . 2 Once tumor size reached 400-500 mm, mice were injected intraperitoneally with 50 mg / kg of GCV every 2-3 days for 2-3 weeks. This treatment regimen resulted in an initial, brief shrinkage of tumors, followed by a tumor size of 400-500 mm after 2-3 weeks of treatment. 2 Tumor size was stabilized by 4% paraformaldehyde. At the end of the experiment, mice were sacrificed and tumors were dissected. A small amount of tissue was snap frozen for RNA extraction, while the remainder was fixed in 4% paraformaldehyde.

[0316] Bioluminescence imaging Mice that developed teratomas derived from cells transfected with the eLuciferase transgene were injected with 30 mg / mL VivoGlo Luciferin at 100 μL / 25 g body weight (Promega #P104C) for 10 minutes and then imaged. Animals were anesthetized with isoflurane and placed in an IVIS Lumina II imager (Caliper Life Sciences) driven by Living Image software. Exposure times ranged from 5 seconds to 5 minutes, depending on signal intensity.

[0317] histology Fixed tumors were embedded in paraffin, sectioned, and stained with hematoxylin / eosin for histological analysis at the CMHD Pathology Core. Histological images were processed using NDPview2 software.

[0318] Example 2: Generation of cloak cells Transgenes encoding the genes in Table 1 were cloned into expression vectors and the sequences were verified by both polymerase chain reaction (PCR), restriction enzyme digestion and sequencing, all using standard methods known in the art.

[0319] A set of constructs (Set 1) containing the transgenes Cd47, Cd200, FasL, and H2-M3 was transfected into mouse embryonic stem cells derived from our C57BL / 6 mouse ES line (C2). The presence of the transgenes was confirmed 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 was transfected into FVB / N-derived ES cells (ES line C2).

[0320] A similar method was used to generate cloaked B16F10 melanoma cells, except that the medium used was DMEM containing 10% fetal bovine serum (FBS).

[0321] Example 3: Screening process for inhibition of T cell activation A modified in vitro mixed lymphocyte reaction (MLR) assay was used to screen for transgene combinations that resulted in the most efficient inhibition of T cell activation. Cell lines transfected with Set 1 and Set 2 cloaking transgenes from Example 1 were used. Donor OT-I 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 with oocyte-expressing cells at a 10:1 ratio. 10,000 of these cells were added to each well of splenocytes. IL-2 was added as a general activator, and T cell proliferation was measured by flow cytometry three days later (Figures 2A-2E). Cells were initially gated to include only CD8+ cells, and all conditions were performed in quadruplicate.

[0322] The negative control (spleen cells only) resulted in a baseline proliferation rate of 6.12% (Figure 2A). Wild-type B16 melanoma (+10% ovarian expression) cells resulted in a clear acceleration of proliferation to 17.1% (Figure 2B), while cloaked cells reduced this proliferation to 9.51% (Figure 2C). Similar results were obtained for wild-type (Figure 2D) versus cloaked ES cells (Figure 2E).

[0323] Example 4: Studies with WT and Cloak cancer cells in syngeneic and allograft B16F10 melanoma cells Because some of the candidate cloaking transgenes are intended to inhibit or modulate the initiation phase of the immune recognition cascade, the effects of these transgenes can be assessed 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.

[0324] This necessitated an alternative assay capable of screening a large number of transgene combinations in an in vivo allogeneic setting. Intraperitoneal and intravenous injection of ES cells with various transgene combinations was attempted as an option. However, the development of teratomas depended on the aggregation of a minimum number of ES cells (1 × 10 depending on the injection site).5 ~5×10 6 ), making this option incompatible with such screening. However, the murine melanoma cancer cell line B16F10, derived from C57BL / 6 mice, is not so limited. 5×10 3 Intravenous injection of less than 100 μg results in the efficient introduction of a wide variety of cancer nodules in the lung. By limiting the number of cells injected, cancer cells can be expected to become trapped in the alveoli and form nodules derived from a single cell or a very small number of cells. These nodules can be isolated and genotyped to identify the transgene.

[0325] Injection of B16F10 melanoma cells into the bloodstream of C57BL / 6 mice (syngraft control) resulted in the formation of cancer nodules in the lungs (Figure 3A, left panel). However, when observed 14 days after injection, small melanoma nodules also formed in the lungs of negative control mice (syngeneic control FVB mice transplanted with wild-type B16F10 melanoma). However, when the melanomas were allowed to grow for 24 days, the nodules almost completely regressed (Figure 3A, right panel).

[0326] We repeated the above experiment by injecting a mixture of cancer cells expressing random combinations of candidate cloaking genes, generated using the PiggyBac transposon system. Lung nodules that developed in an allogeneic setting contained the successful combinations necessary to recognize and protect the allograft from rejection (Figure 3B, right panel). The same immune cloak cells also generated accelerated development in syngeneic hosts (Figure 3B, left panel).

[0327] Example 5: Non-cloaked embryonic stem cells do not form teratomas in an allogeneic setting As shown in Table 4, wild-type ESCs derived from C57BL / 6 mice were confirmed to be unable to form teratomas in FVB / N mice. Similarly, we also demonstrated that wild-type ESCs derived from the FVB / N background were unable to form teratomas in C57BL / 6 hosts. ES cell colonies were dissociated with trypsin, washed once with additive-free DMEM, and resuspended in Matrigel HC at a concentration of approximately 50 million cells per milliliter. Recipient mice were anesthetized, and 100 microliters were injected subcutaneously into each flank area. Developing teratomas were followed for 12 weeks and confirmed by palpation and volume measurement with a vernier caliper.

[0328] [Table 10]

[0329] Example 6: Cloaked ES cells can be propagated in syngeneic and allogeneic hosts To verify the cloaking ability of candidate transgenes, ESCs were transfected with the same transgene and simultaneously added a luciferase transgene, which can be detected by imaging. Briefly, ES cells were prepared as described above. The presence of viable cells was repeatedly measured by imaging. The images in Figure 4 were taken 17 days after injection.

[0330] In Figure 4, the top panel shows the proliferation of immune cloak cells in a syngeneic host, while the bottom panel shows the proliferation of immune cloak cells in an allogeneic host.

[0331] In another experiment, cloned ES cells from C57BL / 6 mice with high expression of eight immunomodulatory transgenes (clone NT2) were subcutaneously injected into different allogeneic mouse strains (C3H, FVB / N, and CD1) with mismatched MHC alleles. Red arrows indicate the teratomas that formed (Figures 5A-5C).

[0332] Example 7: Mice with cloaked tissue are immune intact Non-immune cloak (wild-type) ESCs were transplanted into mice with pre-existing immune cloak tissue, and the mice were evaluated to determine whether they could effectively reject the non-immune cloak graft (Figure 6). The same mice were imaged several times over a 15-day period. As shown in the left panel of Figure 6, in syngeneic control mice, the graft was not rejected over time. In allogeneic FVB mice, the left mouse in the right panel of Figure 6 had a pre-existing immune cloak graft (arrow). The mouse in the center of the right panel of Figure 6 was previously transplanted with C57BL / 6 allogeneic ESCs but rejected the graft (without being bound by theory, the rejection may have been due to pre-formed antibodies against the C57BL / 6 cells). The mouse on the right side of the right panel of Figure 6 had not been previously transplanted. All three mice successfully rejected the non-immune cloak graft. The mouse on the right rejected the graft more slowly, which may have been due to the lack of pre-formed antibodies against the C57BL / 6 cells.

[0333] A similar experiment was performed in which wild-type embryonic stem cells were detected up to 9 days after injection into FVB / N mice bearing cloaked teratomas (Figure 7). However, by day 12, no evidence of residual cells could be detected. Control animals were C57BL / 6 mice also bearing cloaked tumors. The signal in these mice increased over the time course of the experiment.

[0334] Example 8: Cloaking and Fail-Safe Embryonic Stem Cell Lines When a fail-safe C57BL / 6 ES cell line (e.g., as described in WO / 2016 / 141480) was cotransfected with five candidate cloaking transgenes (PD-L1, FasL, Cd47, Cd200, and H2-M3), none of these transgene lines produced teratomas in the allograft setting. When the set of cotransfected genes was expanded with three additional candidate cloaking genes: Spi6, Ccl21b, and Mfge8, 38 clonal lines were generated. One of these lines, NT2, produced teratomas in allogeneic recipients (FVB). The expression levels of the cloaking genes in the 38 clonal lines, including the NT2 line (see arrows in Figure 8A-H), were measured using quantitative PCR (Figure 8A-H). Among the 38 clones, NT2 had the highest overexpression of Ccl21b (16,000x), FasL (25,000x), Cd200 (1700x), Cd47 (16x), Mfge8 (34x), Spi6 (600x), and H2-M3 (750x) compared to WT ES cells. Although PD-L1 was not the highest expresser among the clones, its expression was also significantly increased by 350x in ES cells. The expression of these genes was also checked 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, thus indicating that their relative expression to ES cells is very high. Based on this data, these eight highly activated genes may play a major role in inducing allograft immune tolerance.

[0335] NT2 cells were injected into C57BL / 6 mice to generate teratomas in both the FVB allogeneic setting (Figures 11A-11B) and the FVB syngeneic C57BL / 6 syngeneic setting. Allogeneic teratomas (n=6) grew steadily from day 12 to day 38. 2At a size of 1000 μg / cm, ganciclovir (GCV) treatment was initiated to eliminate the proliferative component of the tumor (Figures 12A-12B, top panel (Figure 12A) syngeneic teratoma; bottom panel (Figure 12B) allogeneic teratoma). 20 days of treatment halted allograft growth. This experiment demonstrates that 1) failsafe and cloak (NT2) cell-derived teratomas respond similarly to GCV treatment, entering a dormant state after brief GCV exposure; 2) after GCV, teratomas remain stable without signs of rejection of the dormant tissue; and 3) the kinetics of teratoma growth in FVB animals differs from that in C57BL / 6.

[0336] Highly expressed cloaking transgenes survive to form teratomas in allogeneic mice. In our system, the cloaking transgene is expressed under the very strong synthetic promoter CAG (shown in the schematic diagram in Figure 19). The CAG promoter is a combination of the cytomegalovirus early enhancer element, the splicer acceptor and promoter of the rabbit β-globin gene, and the first exon and first intron of the chicken β-actin gene. We performed extensive qPCR analysis of the transgene transcript levels in many different ES cell clones, each with different transgene expression levels. Only the ES clones with the highest expression of the cloaking transgene survive in allogeneic hosts.

[0337] As shown in Figure 9, transcript expression levels of immunomodulatory genes associated with the cloaking technique varied among ES cell clones. Concentric circles are plotted on a log10 scale. The thick black line represents 1x, the next outer ring represents 10x, and then 100x. The innermost ring represents 0.1x. All values ​​are normalized to a positive control, activated leukocytes isolated from mouse lymphoid organs that naturally express the immunomodulatory transgene. The upper left panel shows wild-type ES cells without transgenic modification for reference; they express little or no relevant immunomodulatory transgene. In contrast, clone NT2 and clone 15 (indicated by a red square) both had high expression of the gene and survived in allogeneic hosts. All other clones shown in Figure 9 did not survive in allogeneic hosts.

[0338] The high expression of cloaking transgenes is also shown in Figure 10. As shown in Figure 10, all eight cloaking transgenes in the NT2 cell line and NT2-derived teratomas have expression levels in the top 5% of all genes in the ES cell genome, and five of the cloaking transgenes have expression levels in the top 1% of all genes in the ES cell genome. Because only one of these genes has an expression level in the top 5% of all genes in the genome, the expression of these genes is much lower in WT ES cells.

[0339] Example 9: Cloaked ESCs contribute to all three germ layers in allogeneic teratomas We next asked whether immune cloaking could extend the full pluripotent developmental potential of ESCs in teratomas. Teratomas resulting from the injection of cloaked and uncloned ESCs derived from C57BL / 6 mice into syngeneic and allogeneic hosts were analyzed by histopathology (hematoxylin and eosin staining). Figures 13A-13B (syngeneic host neurons, bone, and columnar epithelium in the upper panel (Figure 13A); and allogeneic host neurons, bone, columnar epithelium, and blood vessels in the lower panel (Figure 13B)) show representative images from both backgrounds, demonstrating that expression of the cloaking transgene does not interfere with the normal developmental potential of these ES cells and that tumors are well vascularized. Both syngeneic and allogeneic tissues showed no immune cell infiltration.

[0340] In another experiment, we tested whether the cloned ES cells were truly pluripotent by examining whether they could form cells from all three germ layers (endoderm, ectoderm, and mesoderm) (Figures 14A-14D). 6 ~10 7 The assay involved injecting cloaked ES cells subcutaneously into mice and allowing them to proliferate and differentiate into tissue masses called teratomas. The teratomas were then removed 3-4 weeks after ES cell injection, and tissue sections were cut and stained with H&E. These sections were analyzed under a microscope for cell morphology to determine whether all three germ layers were present.

[0341] We asked whether eight cloaking transgenes inserted into ES cells and expressed at high levels disrupted their ability to form all three germ layers. They did not. Figures 14A-14C show the three germ layers (ec = ectoderm, shown in Figure 14A; en = endoderm, shown in Figure 14C; me = mesoderm, shown in Figure 14B). Figure 14D shows blood vessels verifying that these tissues are well vascularized.

[0342] Example 10: ES cells expressing a cloaking transgene produce the protein encoded by the transgene We directly confirmed the presence of the protein encoded by the cloaking transgene in NT2 ES cells (one of the clones with the highest expression) using fluorescent antibody-based microscopy (Figures 16A-16H). These data confirm that the protein encoded by the transgene is expressed in ES cells at readily detectable levels, as expected based on the high level of mRNA expression.

[0343] Example 11: ES cells expressing high levels of a cloaking transgene have typical morphology and express common ES cell markers We analyzed the cloaked ES cells to determine whether they expressed ES cell markers and retained normal ES cell morphology. Cloaked ES cells possessed the typical morphology observed in healthy pluripotent ES cells (Figure 17A) and stained positive for alkaline phosphatase, a hallmark of healthy pluripotent ES cells (Figure 17B). Furthermore, our cloaked ES cells stained positive for the transcription factors Oct4 (Figure 18A) and SSEA (Figure 18B), both common markers of normal pluripotent ES cells, using fluorescent antibodies. These data demonstrate that ES cells expressing high levels of eight immunomodulatory cloning transgenes appear as normal ES cells with respect to their morphology and expression of common ES cell markers. The insets show that staining for Oct4 and SSEA1 (bottom left inset) colocalizes with ES cells (visualized using DAPI in the top right inset).

[0344] Example 12: IFNγR1 d39 prevents MHC upregulation in ES cells Activated T cells secrete IFNγ, which binds to the IFNγR1 / R2 complex expressed on many cell types, including tissues and cells derived from ES cells. IFNγ binding to the IFNγ receptor induces upregulation of HLA (MHC in mice) and HLA-associated molecules on the cell surface, which increases the likelihood of allograft allogeneic and immune rejection. Differences in HLA proteins (also called major antigens) between donor and recipient are the primary cause of rejection in allogeneic transplants.

[0345] To assess whether disruption of IFNγ signaling inhibits or reduces HLA upregulation, we transfected C57BL / 6 ES cells with piggyBac integratable vectors containing wild-type IFNγR1 or dominant-negative IFNγR1 (IFNγR1 d39, lacking 39 amino acids in the cytoplasmic tail) transgenes, which were expressed under the control of a constitutive CAG promoter upstream of the transgene contained on the same piggyBac integration cassette.

[0346] Wild-type and transfected ES cells were then grown in culture and exposed to 100 ng / mL of IFNγ ligand for 24 hours. In wild-type ES and IFNγR1-transfected cells (left and center panels of Figure 20, respectively), IFNγ exposure increased H-2k b and H-2D bThis resulted in increased expression of major histocompatibility surface molecules (MHC class I) in IFNγR1 d39 cells, but not in IFNγR1 d39 cells (right panel of Figure 20). Exposure to PBS alone had no effect. MHC class I levels were detected by fluorescent antibody staining, and expression levels were quantified by ...

Claims

1. Cells genetically modified to contain at least one mechanism for providing local immunosuppression at the transplant site when transplanted into an allogeneic host, - a set of transgenes, each transgene being a cytoplasmic, membrane-bound or locally acting gene product, the function of which is to a) attenuating the activation and function of antigen-presenting cells; b) attenuating graft-attacking leukocyte activity or cytolytic function; c) attenuating macrophage cytolytic function and phagocytosis of allograft cells; d) inducing apoptosis in graft-attacking leukocytes; e) mitigating local inflammatory proteins; and f) Protecting against leukocyte-mediated apoptosis a set of transgenes encoding gene products that are one or more of wherein the set of transgenes comprises 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, or genes encoding a biological substance that acts as an agonist of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, or Serpin B9 or Spi6.

2. 2. The cell of claim 1, wherein the set of transgenes comprises three, four, five, six, seven, or all eight 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, or genes encoding biological substances that act as agonists of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, or Serpin B9 or Spi6.

3. 2. The cell of claim 1, wherein the set of transgene genes comprises genes encoding PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6, or genes encoding a biological substance that acts as an agonist of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6.

4. 4. The cell of any one of claims 1 to 3, further comprising one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNγR1 d39 or a gene encoding a biological substance that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 or IFNγR1 d39.

5. The method of claim 4, wherein the TGF-β or biological substance acts locally within the graft environment.

6. 6. The cell of any one of claims 1 to 5, which is a source of stem cells, cells amenable to genome editing and / or therapeutic cell types.

7. Embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germline stem cells, lung stem cells or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte precursor cells, B cells, exocrine cells, pancreatic precursor cells, endocrine precursor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, bone cells, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, natural killer cells, skeletal 7. The cell of claim 6, which is a muscle cell, a cardiac muscle cell, an erythroid-megakaryocytic cell, an eosinophil, a macrophage, a T cell, an islet beta cell, a neuron, a cardiac muscle cell, a blood cell, 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, a white or brown adipocyte, a hormone-secreting cell, an epidermal keratinocyte, an epithelial cell, a kidney cell, a germ cell, a skeletal joint synoviocyte, a periosteal cell, a perichondrocyte, a chondrocyte, 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, a serous cell, a somatic cell, or a cell derived from the skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.

8. further comprising at least one mechanism for controlling cell proliferation; a) genetic modification of one or more cell division loci (CDLs), wherein the CDLs are one or more endogenous or exogenous loci whose transcription products are expressed by dividing cells, the genetic modification comprising: i) an Ablation Link (ALINK) system comprising a DNA sequence encoding a negative selection marker that is transcriptionally linked to a DNA sequence encoding the CDL; and ii) an exogenous activator of regulation (EARC) system for CDLs, comprising an inducible activator-based gene expression system operably linked to said CDLs; Genetic modification, which is one or more of The cell according to any one of claims 1 to 7, comprising:

9. The genetic modification of the CDL comprises: 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 9. The cell of claim 8, comprising targeted replacement of the CDL with one or more of the ALINK and / or EARC systems, each operably linked to the CDL.

10. The cell of claim 8 or 9, 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 results in activation of the CDL only by an inducer of the inducible activator-based gene expression system.

11. 11. The cell of any one of claims 8 to 10, wherein the CDL is one or more of the loci listed in Table 5.

12. 12. The cell of claim 11, wherein the CDL encodes a gene product that functions in one or more of the cell cycle, DNA replication, RNA transcription, protein translation, and metabolism.

13. 13. The cell of claim 12, wherein 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.

14. The cell according to any one of claims 8 to 13, 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, and preferably the ALINK system is a herpes simplex virus-thymidine kinase / ganciclovir system.

15. The cell according to any one of claims 8 to 13, wherein the EARC system is a dox-crosslinking system, a cumate switch induction system, an ecdysone induction system, an electromagnetic wave induction system, or a ligand reversible dimerization system, and preferably the EARC system is a dox-crosslinking system.

16. 16. The cell of any one of claims 1 to 15, wherein one or more 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 equal to or greater than the expression level of the corresponding endogenous gene in activated leukocytes.

17. The cell of claim 16, 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 levels equal to or greater than the expression levels of the corresponding endogenous genes in activated leukocytes.

18. 18. The cell of any one of claims 1 to 17, wherein the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:

12.

19. The cell of any one of claims 1 to 18, wherein the HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO:

15.

20. 20. The cell of any one of claims 1 to 19, wherein the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

21. The cell of any one of claims 1 to 20, wherein the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:

6.

22. The cell of any one of claims 1 to 21, wherein the FASLG or FasL transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:

9.

23. 23. The cell of any one of claims 1 to 22, wherein the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

1.

24. 24. The cell of any one of claims 1 to 23, wherein the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:

14.

25. The cell of any one of claims 1 to 24, wherein the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:

7.

26. The cell of any one of claims 4 to 25, wherein the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:

17.

27. The cell of any one of claims 1 to 26, wherein the two or more transgenes are operably linked to a constitutive promoter.

28. The cell of claim 27, 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

29. The cell of any one of claims 1 to 28, further comprising a transgene encoding a therapeutic agent.

30. 30. The cell of claim 29, wherein the therapeutic agent is a protein or an antibody.

31. The cell of claim 30 , wherein the antibody is an inhibitory antibody or an agonist antibody.

32. 32. The cell of any one of claims 29-31, wherein the therapeutic agent is a drug listed in Table 2 or a wild-type version of a gene known to be mutated in cancer, enzyme or hormone deficiency, metabolic disease, or degenerative disease.

33. 33. The cell of any one of claims 29 to 32, wherein 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 radiogenic system, a cumate switch-inducible system, an ecdysone-inducible system, a destabilization domain system, or a ligand-reversible dimerization system.

34. The cell of any one of claims 29 to 32, wherein the therapeutic agent is expressed using a constitutive promoter 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

35. A population of genetically modified cells comprising the cells of any one of claims 1 to 34.

36. 36. A method for providing local immunosuppression at a transplant site in an allogeneic host, the method comprising transplanting a population of cells according to any one of claims 1 to 34 or genetically modified cells according to claim 35 into the transplant site in the allogeneic host.

37. 1. A method for providing local immunosuppression at a transplant site in an allogeneic host, comprising: (i) providing a cell; and (ii) expressing a set of transgenes in said cell, each transgene being a cytoplasmic, membrane-bound or locally acting gene product, the function of which is to a) attenuating the activation and function of antigen-presenting cells; b) attenuating graft-attacking leukocyte activity or cytolytic function; c) attenuating macrophage cytolytic function and phagocytosis of allograft cells; d) inducing apoptosis in graft-attacking leukocytes; e) mitigating local inflammatory proteins; and f) Protecting against leukocyte-mediated apoptosis encoding or expressing a gene product, which is one or more of: wherein the set of transgenes comprises two or more of the following genes: PD-L1, HLAG or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6, or genes encoding biological substances that act as agonists of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, or Serpin B9 or Spi6.

38. 38. The method of claim 37, wherein the set of transgenes comprises three, four, five, six, seven, or all eight 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, or genes encoding biological substances that act as agonists of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, or Serpin B9 or Spi6.

39. 38. The method of claim 37, wherein the set of transgene genes comprises genes encoding PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6, or genes encoding a biological substance that acts as an agonist of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6.

40. 40. The method of any one of claims 37 to 39, further comprising expressing one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNγR1 d39 or a gene encoding a biological substance that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 or IFNγR1 d39.

41. 41. The method of claim 40, wherein the TGF-β or biological agent acts locally within the graft environment.

42. 42. The method of any one of claims 37 to 41, wherein the cells are stem cells, cells amenable to genome editing and / or a source of therapeutic cell types.

43. The cells may be embryonic stem cells, induced pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germline stem cells, lung stem cells or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte progenitor cells, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, osteocytes, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, natural killer cells, 43. The method of claim 42, wherein the cell is a cell selected from the group consisting of: a cytoplasmic reticulum, a skeletal muscle cell, a cardiac muscle cell, an erythroid-megakaryocyte cell, an eosinophil, a macrophage, a T cell, an islet beta cell, a neuron, a cardiac muscle cell, a blood cell, 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, a white or brown adipocyte, a hormone-secreting cell, an epidermal keratinocyte, an epithelial cell, a kidney cell, a germ cell, a skeletal joint synoviocyte, a periosteal cell, a perichondrocyte, a chondrocyte, 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, a serous cell, a somatic cell, or a cell derived from the skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.

44. A method for controlling proliferation of the cells according to any one of claims 1 to 7 at a transplant site in an allogeneic host, comprising: a) genetically modifying a cell division locus (CDL) in a cell, said CDL being one or more endogenous or exogenous loci whose transcription products are expressed by dividing cells, said genetic modification of said CDL comprising: i) an Ablation Link (ALINK) system comprising a DNA sequence encoding a negative selection marker that is transcriptionally linked to a DNA sequence encoding the CDL; and ii) an inducible exogenous activator of regulation (EARC) system of a CDL, comprising an inducible activator-based gene expression system operably linked to said CDL. Genetically modifying the gene, including one or more of: b) transplanting said cell or said population of cells into a transplantation site in an allogeneic host; and c) i) maintaining the genetically modified cells comprising the ALINK system in the absence of an inducer of the negative selection marker, thereby allowing proliferation of the genetically modified cells comprising the ALINK system, and / or ablating or inhibiting proliferation of the genetically modified cells comprising the ALINK system, by exposing the cells comprising the ALINK system to the inducer of the negative selection marker; and / or ii) exposing the genetically modified cells containing the EARC system to an inducer of the inducible activator-based gene expression system, thereby allowing proliferation of the genetically modified cells containing the EARC system, or maintaining the cells containing the EARC system in the absence of the inducer of the inducible activator-based gene expression system, thereby preventing or inhibiting proliferation of the genetically modified cells containing the EARC system. A method comprising:

45. The method of any one of claims 36 to 44, wherein the homologous host is a mammal.

46. 46. ​​The method of claim 45, wherein the allogeneic host is a mouse.

47. 46. ​​The method of claim 45, wherein the allogeneic host is a human.

48. 48. The method of any one of claims 36 to 47, wherein the host has a degenerative disease or condition that can be treated using cell therapy.

49. 49. The method of claim 48, wherein 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, enzyme or hormone deficiency, metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infectious disease, hemophilia, myocardial infarction, degenerative disease or age-related disease.

50. 50. The method of any one of claims 37 to 49, wherein one or more 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 equal to or greater than the expression level of the corresponding endogenous gene in activated leukocytes.

51. 51. The method of claim 50, 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 levels equal to or greater than the expression levels of the corresponding endogenous genes in activated leukocytes.

52. 52. The method of any one of claims 37 to 51, wherein the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:

12.

53. 53. The method of any one of claims 37 to 52, wherein the HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO:

15.

54. 54. The method of any one of claims 37 to 53, wherein the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

55. The method of any one of claims 37 to 54, wherein the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:

6.

56. 56. The method of any one of claims 37 to 55, wherein the FASLG or FasL transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:

9.

57. 57. The method of any one of claims 37 to 56, wherein the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

1.

58. 58. The method of any one of claims 37 to 57, wherein the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:

14.

59. The method of any one of claims 37 to 58, wherein the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:

9.

60. 60. The method of any one of claims 40 to 59, wherein the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:

17.

61. 61. The method of any one of claims 37 to 60, wherein the one or more transgenes are operably linked to a constitutive promoter.

62. 62. The method of claim 61, 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

63. 63. The method of any one of claims 37 to 62, wherein the cells further comprise a transgene encoding a therapeutic agent.

64. 64. The method of claim 63, wherein the therapeutic agent is a protein or an antibody.

65. 65. The method of claim 64, wherein the antibody is an inhibitory antibody or an agonist antibody.

66. 66. The method of any one of claims 63-65, wherein the therapeutic agent is an agent listed in Table 2 or a wild-type version of the gene that is mutated in the subject.

67. 67. The method of any one of claims 63 to 66, wherein 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 radiogenic system, a cumate switch-inducible system, an ecdysone-inducible system, a destabilization domain system, or a ligand-reversible dimerization system.

68. 67. The method of any one of claims 63 to 66, wherein the therapeutic agent is expressed using a constitutive promoter 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

69. A composition comprising the cells of any one of claims 1 to 34.

70. 70. The composition of claim 69, further comprising a pharmaceutically acceptable excipient.

71. A kit comprising a cell according to any one of claims 1 to 34 or a composition according to claim 69 or 70.

72. 71. A method of treating a disease or condition in a subject in need thereof, comprising administering to said subject a cell according to any one of claims 1 to 34 or a composition according to claim 69 or 70.

73. 73. The method of claim 72, wherein the disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, an enzyme or hormone deficiency, a metabolic disease, an autoimmune disease, age-related macular degeneration, retinal dystrophy, an infectious disease, hemophilia, myocardial infarction, a degenerative disease, an age-related disease, or a disease or condition listed in Table 2.

74. 74. The method of claim 72 or 73, wherein the cells are differentiated into a lineage-restricted cell type prior to administration to the subject.

75. 75. The method of claim 74, wherein the disease or condition is myocardial infarction and the cells are differentiated into cardiomyocytes.

76. 75. The method of claim 74, wherein the disease or condition is blindness and the cells are differentiated into photoreceptor cells.

77. 75. The method of claim 74, wherein the disease or condition is spinal cord injury, Parkinson's disease, Huntington's disease, or Alzheimer's disease, and the cells are differentiated into neurons.

78. 75. The method of claim 74, wherein the disease or condition is multiple sclerosis and the cells are differentiated into glial cells.

79. 79. The method of any one of claims 72 to 78, wherein the cells are administered locally to a tissue or body site in need of cells or the therapeutic agent.

80. 80. The method of any one of claims 72 to 79, wherein the cells are administered intravenously, subcutaneously, intramuscularly, transcutaneously, intradermally, parenterally, intraarterially, intravascularly or by perfusion.

81. 81. The method of claim 80, wherein the cells are administered by subcutaneous injection to produce cloaked subcutaneous tissue.

82. 80. The method of any one of claims 72 to 79, wherein the cells are administered as a tissue.

83. 83. The method of claim 82, wherein the tissue is administered with a gel, a biocompatible matrix, or a cell scaffold.

84. 80. The method of any one of claims 72 to 79, wherein the cells are administered in an amount of 25,000 to 5,000,000,000 cells.

85. 84. The method of any one of claims 80 to 83, wherein the cells are administered in an amount of 800,000,000 to 3,000,000,000 cells.

86. 86. The method of any one of claims 72 to 85, further comprising administering an additional therapeutic agent.

87. 87. The method of claim 86, wherein the additional therapeutic agent is administered prior to administration of the cells.

88. 87. The method of claim 86, wherein the additional therapeutic agent is administered after administration of the cells.

89. 87. The method of claim 86, wherein the additional therapeutic agent is administered simultaneously with administration of the cells.

90. The additional therapeutic agent may be an immunosuppressant, a disease-modifying antirheumatic drug (DMARD), a biological 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, kinelet, certolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab, 6 -mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab, aminosalicylate, antibiotic, antihistamine, anti-TNFα, azathioprine, belimumab, beta interferon, calcineurin inhibitor, certolizumab, corticosteroid, cromolyn, cyclosporine A, cyclosporine, dimethyl fumarate, etanercept, fingolimod, fumarate ester, glatiramer acetate, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonist, long-acting beta β2 agonists, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, probiotics, retinoids, salicylic acid, short-acting β2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab or vedolizumab, bevacuzimab, ranibizumab or aflibercept), photodynamic therapy, photocoagulation, carbidopa-levodopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase inhibitors, anticholinergics, amoxicillin angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, angiotensin receptor neprilysin inhibitors, beta blockers, combination alpha and beta blockers, calcium channel blockers, cholesterol lowering drugs, nicotinic acid, cholesterol absorption inhibitors, digitalis preparations, diuretics, vasodilators, dual antiplatelet therapy, cardiac procedures, antiviral compounds, nucleoside analog reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, antibacterial compounds, antifungal compounds,90. The method of any one of claims 86 to 89, wherein the therapeutic agent is an antiparasitic compound, insulin, a sulfonylurea, a biguanide, a meglitinide, a thiazolidinedione, a DPP-4 inhibitor, an SGLT2 inhibitor, an α-glucosidase inhibitor, a bile acid sequestrant, aspirin, dietary therapy, a clotting factor, desmopressin, a clot-preserving agent, a fibrin sealant, physical therapy, a coenzyme, a bone marrow transplant, an organ transplant, hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, a medium-chain triacylglycerol, miglustat, an enzyme replacement therapy, a checkpoint inhibitor, a chemotherapeutic agent, a biological agent, radiation therapy, cryotherapy, hyperthermia, surgical resection or tumor tissue, or an anti-cancer vaccine.

91. 91. The method of any one of claims 72 to 90, further comprising controlling proliferation of the cells.

92. The cells comprise an ALINK system, and the method of controlling proliferation comprises: i) maintaining the cells containing the ALINK system in the absence of an inducer of the negative selection marker, thereby allowing the cells containing the ALINK system to proliferate; or ii) ablating or inhibiting the proliferation of the cells containing the ALINK system by exposing the cells containing the ALINK system to the inducer of the negative selection marker.

92. The method of claim 91, comprising:

93. The cells comprise an EARC system, and the method of controlling proliferation comprises: i) exposing the cells containing the EARC system to an inducer of the inducible activator-based gene expression system, thereby allowing the cells containing the EARC system to proliferate; or ii) preventing or inhibiting proliferation of the cells containing the EARC system by maintaining the cells containing the EARC system in the absence of the inducer of the inducible activator-based gene expression system.

92. The method of claim 91, comprising:

94. 94. The method of any one of claims 72 to 93, wherein the cells are removed after the treatment is completed.

95. A cell according to any one of claims 1 to 34 or a composition according to claim 69 or 70 for use in treating a disease or condition in a subject in need thereof.

96. 96. The cell of claim 95, wherein the disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, an enzyme or hormone deficiency, a metabolic disease, an autoimmune disease, age-related macular degeneration, retinal dystrophy, an infectious disease, hemophilia, myocardial infarction, a degenerative disease, an age-related disease, or a disease or condition listed in Table 2.

97. 71. A cell according to any one of claims 1 to 34 or a composition according to claim 69 or 70 for use in providing local immunosuppression at the site of transplantation in an allogeneic host.

98. 2. The cell of claim 1, further comprising one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or a gene encoding a biological substance that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.

99. The cell of claim 98, wherein the TGF-β or biological substance acts locally within the graft environment.

100. 10. The cell of claim 1, which is a stem cell, a cell amenable to genome editing, and / or a source of therapeutic cell types.

101. Embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germline stem cells, lung stem cells or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte precursor cells, B cells, exocrine cells, pancreatic precursor cells, endocrine precursor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, bone cells, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, natural killer cells, skeletal muscle 101. The cell of claim 100, which is a cell, a cardiac muscle cell, an erythroid-megakaryocytic cell, an eosinophil, a macrophage, a T cell, an islet beta cell, a neuron, a cardiomyocyte, a blood cell, 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, a white or brown adipocyte, a hormone-secreting cell, an epidermal keratinocyte, an epithelial cell, a kidney cell, a germ cell, a skeletal joint synoviocyte, a periosteal cell, a perichondrocyte, a chondrocyte, 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, a serous cell, a somatic cell, or a cell derived from the skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.

102. further comprising at least one mechanism for controlling cell proliferation; a) genetic modification of one or more cell division loci (CDLs), wherein the CDLs are one or more endogenous or exogenous loci whose transcription products are expressed by dividing cells, the genetic modification comprising: i) an Ablation Link (ALINK) system comprising a DNA sequence encoding a negative selection marker that is transcriptionally linked to a DNA sequence encoding the CDL; and ii) an exogenous activator of regulation (EARC) system for CDLs, comprising an inducible activator-based gene expression system operably linked to said CDLs; Genetic modification, which is one or more of The cell of claim 1 , comprising:

103. The genetic modification of the CDL comprises: 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 103. The cell of claim 102, comprising targeted replacement of the CDL with one or more of the ALINK and / or EARC systems, each operably linked to the CDL.

104. The cell of claim 102, 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 results in activation of the CDL only by an inducer of the inducible activator-based gene expression system.

105. 103. The cell of claim 102, wherein the CDL is one or more of the loci listed in Table 5.

106. 106. The cell of claim 105, wherein the CDL encodes a gene product that functions in one or more of the cell cycle, DNA replication, RNA transcription, protein translation, and metabolism.

107. 107. The cell of claim 106, wherein the CDL is one or more of Cdkl / CDKl, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5 and Eef2 / EEF2, preferably wherein the CDL is Cdkl or CDKl.

108. The cell according to claim 102, 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, and preferably the ALINK system is a herpes simplex virus-thymidine kinase / ganciclovir system.

109. The cell described in claim 102, wherein the EARC system is a dox-crosslinking system, a cumate switch induction system, an ecdysone induction system, an electromagnetic wave induction system, or a ligand reversible dimerization system, and preferably the EARC system is a dox-crosslinking system.

110. 2. The cell of claim 1, wherein one or more 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 equal to or greater than the expression level of the corresponding endogenous gene in activated leukocytes.

111. The cell of claim 110, 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 levels equal to or greater than the expression levels of the corresponding endogenous genes in activated leukocytes.

112. 2. The cell of claim 1, wherein the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:

12.

113. The cell of claim 1, wherein the HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO:

15.

114. 2. The cell of claim 1, wherein the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

115. The cell of claim 1, wherein the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:

6.

116. The cell of claim 1 , wherein the FASLG or FasL transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:

9.

117. The cell of claim 1 , wherein the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

1.

118. The cell of claim 1, wherein the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:

14.

119. The cell of claim 1 , wherein the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:

7.

120. 5. The cell of claim 4, wherein the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:

17.

121. The cell of claim 1 , wherein the two or more transgenes are operably linked to a constitutive promoter.

122. The cell of claim 121, 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

123. The cell of claim 1 , further comprising a transgene encoding a therapeutic agent.

124. The cell of claim 123, wherein the therapeutic agent is a protein or an antibody.

125. The cell of claim 124, wherein the antibody is an inhibitory antibody or an agonist antibody.

126. 124. The cell of claim 123, wherein the therapeutic agent is a drug listed in Table 2 or a wild-type version of a gene known to be mutated in cancer, enzyme or hormone deficiency, metabolic disease, or degenerative disease.

127. The cell of claim 123, wherein 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 radiogenic system, a cumate switch-inducible system, an ecdysone-inducible system, a destabilization domain system, or a ligand-reversible dimerization system.

128. The cell of claim 123, wherein the therapeutic agent is expressed using a constitutive promoter 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

129. A population of genetically modified cells comprising the cells of claim 1.

130. A method for providing local immunosuppression at a transplant site in an allogeneic host, comprising transplanting a population of cells described in claim 1 or genetically modified cells described in claim 129 into the transplant site in the allogeneic host.

131. 38. The method of claim 37, further comprising expressing one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or a gene encoding a biological agent that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.

132. 132. The method of claim 131, wherein the TGF-β or biological agent acts locally within the graft environment.

133. 38. The method of claim 37, wherein the cells are stem cells, cells amenable to genome editing and / or a source of therapeutic cell types.

134. The cells include embryonic stem cells, induced pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germline stem cells, lung stem cells or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte precursor cells, B cells, exocrine cells, pancreatic precursor cells, endocrine precursor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, osteocytes, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, and natural killer cells. , skeletal muscle cells, cardiac muscle cells, erythroid-megakaryocyte cells, eosinophils, macrophages, T cells, islet 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 adipocytes, hormone-secreting cells, epidermal keratinocytes, epithelial cells, kidney cells, germ cells, skeletal joint synoviocytes, periosteal cells, perichondrocytes, 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, serous cells, somatic cells or cells derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach.

135. 10. A method for controlling proliferation of the cells of claim 1 at a transplant site in an allogeneic host, comprising: a) genetically modifying a cell division locus (CDL) in a cell, said CDL being one or more endogenous or exogenous loci whose transcription products are expressed by dividing cells, said genetic modification of said CDL comprising: i) an Ablation Link (ALINK) system comprising a DNA sequence encoding a negative selection marker that is transcriptionally linked to a DNA sequence encoding the CDL; and ii) an inducible exogenous activator of regulation (EARC) system of a CDL, comprising an inducible activator-based gene expression system operably linked to said CDL. Genetically modifying the gene, including one or more of: b) transplanting said cell or said population of cells into a transplantation site in an allogeneic host; and c) i) maintaining the genetically modified cells comprising the ALINK system in the absence of an inducer of the negative selection marker, thereby allowing proliferation of the genetically modified cells comprising the ALINK system, and / or ablating and / or inhibiting proliferation of the genetically modified cells comprising the ALINK system, by exposing the cells comprising the ALINK system to the inducer of the negative selection marker; and / or ii) exposing the genetically modified cells containing the EARC system to an inducer of the inducible activator-based gene expression system, thereby allowing proliferation of the genetically modified cells containing the EARC system, or maintaining the cells containing the EARC system in the absence of the inducer of the inducible activator-based gene expression system, thereby preventing or inhibiting proliferation of the genetically modified cells containing the EARC system. A method comprising:

136. 38. The method of claim 37, wherein the allogeneic host is a mammal.

137. 137. The method of claim 136, wherein the allogeneic host is a mouse.

138. 137. The method of claim 136, wherein the allogeneic host is a human.

139. 38. The method of claim 37, wherein the host has a degenerative disease or condition that can be treated using cell therapy.

140. 140. The method of claim 139, wherein 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, enzyme or hormone deficiency, metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infectious disease, hemophilia, myocardial infarction, degenerative disease or age-related disease.

141. 38. The method of claim 37, wherein one or more 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 equal to or greater than the expression level of the corresponding endogenous gene in activated leukocytes.

142. 142. The method of claim 141, 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 levels equal to or greater than the expression levels of the corresponding endogenous genes in activated leukocytes.

143. 38. The method of claim 37, wherein the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:

12.

144. 38. The method of claim 37, wherein the HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO:

15.

145. 38. The method of claim 37, wherein the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

4.

146. 38. The method of claim 37, wherein the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:

6.

147. 38. The method of claim 37, wherein the FASLG or FasL transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:

9.

148. 38. The method of claim 37, wherein the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

1.

149. 38. The method of claim 37, wherein the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO:

14.

150. 38. The method of claim 37, wherein the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:

9.

151. 38. The method of claim 37, wherein the IFNγRl d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:

17.

152. 38. The method of claim 37, wherein the one or more transgenes are operably linked to a constitutive promoter.

153. The method of claim 152, 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

154. 38. The method of claim 37, wherein the cell further comprises a transgene encoding a therapeutic agent.

155. 155. The method of claim 154, wherein the therapeutic agent is a protein or an antibody.

156. 156. The method of claim 155, wherein the antibody is an inhibitory antibody or an agonist antibody.

157. 155. The method of claim 154, wherein the therapeutic agent is an agent listed in Table 2 or a wild-type version of the gene that is mutated in the subject.

158. The method of claim 154, wherein 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 radiogenic system, a cumate switch-inducible system, an ecdysone-inducible system, a destabilization domain system, or a ligand-reversible dimerization system.

159. The method of claim 154, wherein the therapeutic agent is expressed using a constitutive promoter 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, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, and a Rous sarcoma virus (RSV) promoter.

160. A composition comprising the cells of claim 1.

161. 161. The composition of claim 160, further comprising a pharmaceutically acceptable excipient.

162. 161. A kit comprising the cell of claim 1 or the composition of claim 160.

163. A method for treating a disease or condition in a subject in need thereof, comprising administering to the subject a cell described in claim 1 or a composition described in claim 160.

164. 164. The method of claim 163, wherein the disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, an enzyme or hormone deficiency, a metabolic disease, an autoimmune disease, age-related macular degeneration, retinal dystrophy, an infectious disease, hemophilia, myocardial infarction, a degenerative disease, an age-related disease, or a disease or condition listed in Table 2.

165. 164. The method of claim 163, wherein the cells are differentiated into a lineage-restricted cell type prior to administration to the subject.

166. 166. The method of claim 165, wherein the disease or condition is myocardial infarction and the cells are differentiated into cardiomyocytes.

167. 166. The method of claim 165, wherein the disease or condition is blindness and the cells are differentiated into photoreceptor cells.

168. 166. The method of claim 165, wherein the disease or condition is spinal cord injury, Parkinson's disease, Huntington's disease or Alzheimer's disease, and the cells are differentiated into neurons.

169. 166. The method of claim 165, wherein the disease or condition is multiple sclerosis and the cells are differentiated into glial cells.

170. 164. The method of claim 163, wherein the cells are administered locally to a tissue or body site in need of the cells or the therapeutic agent.

171. 164. The method of claim 163, wherein the cells are administered intravenously, subcutaneously, intramuscularly, transdermally, intradermally, parenterally, intraarterially, intravascularly, or by perfusion.

172. 172. The method of claim 171, wherein the cells are administered by subcutaneous injection to produce cloaked subcutaneous tissue.

173. 164. The method of claim 163, wherein the cells are administered as a tissue.

174. 174. The method of claim 173, wherein the tissue is administered with a gel, a biocompatible matrix, or a cell scaffold.

175. 164. The method of claim 163, wherein the cells are administered in an amount of 25,000 to 5,000,000,000 cells.

176. 172. The method of claim 171, wherein the cells are administered in an amount of 800,000,000 to 3,000,000,000 cells.

177. 164. The method of claim 163, further comprising administering an additional therapeutic agent.

178. 178. The method of claim 177, wherein the additional therapeutic agent is administered prior to administration of the cells.

179. 178. The method of claim 177, wherein the additional therapeutic agent is administered after administration of the cells.

180. 178. The method of claim 177, wherein the additional therapeutic agent is administered simultaneously with administration of the cells.

181. The additional therapeutic agent may be an immunosuppressant, a disease-modifying antirheumatic drug (DMARD), a biological 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, kinelet, certolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab, 6 -mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab, aminosalicylate, antibiotic, antihistamine, anti-TNFα, azathioprine, belimumab, beta interferon, calcineurin inhibitor, certolizumab, corticosteroid, cromolyn, cyclosporine A, cyclosporine, dimethyl fumarate, etanercept, fingolimod, fumarate ester, glatiramer acetate, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonist, long-acting beta β2 agonists, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, probiotics, retinoids, salicylic acid, short-acting β2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab or vedolizumab, bevacuzimab, ranibizumab or aflibercept), photodynamic therapy, photocoagulation, carbidopa-levodopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase inhibitors, anticholinergics, amoxicillin angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, angiotensin receptor neprilysin inhibitors, beta blockers, combination alpha and beta blockers, calcium channel blockers, cholesterol lowering drugs, nicotinic acid, cholesterol absorption inhibitors, digitalis preparations, diuretics, vasodilators, dual antiplatelet therapy, cardiac procedures, antiviral compounds, nucleoside analog reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, antibacterial compounds, antifungal compounds,178. The method of claim 177, which is an antiparasitic compound, insulin, a sulfonylurea, a biguanide, a meglitinide, a thiazolidinedione, a DPP-4 inhibitor, an SGLT2 inhibitor, an α-glucosidase inhibitor, a bile acid sequestrant, aspirin, dietary therapy, a clotting factor, desmopressin, a clot-preserving agent, a fibrin sealant, physical therapy, a coenzyme, a bone marrow transplant, an organ transplant, hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, a medium-chain triacylglycerol, miglustat, an enzyme replacement therapy, a checkpoint inhibitor, a chemotherapeutic agent, a biological agent, radiation therapy, cryotherapy, hyperthermia, surgical resection or tumor tissue, or an anti-cancer vaccine.

182. 164. The method of claim 163, further comprising controlling proliferation of the cells.

183. The cells comprise an ALINK system, and the method of controlling proliferation comprises: i) maintaining the cells containing the ALINK system in the absence of an inducer of the negative selection marker, thereby allowing the cells containing the ALINK system to proliferate; or ii) ablating or inhibiting the proliferation of the cells containing the ALINK system by exposing the cells containing the ALINK system to the inducer of the negative selection marker.

183. The method of claim 182, comprising:

184. The cells comprise an EARC system, and the method of controlling proliferation comprises: i) exposing the cells containing the EARC system to an inducer of the inducible activator-based gene expression system, thereby allowing the cells containing the EARC system to proliferate; or ii) preventing or inhibiting proliferation of the cells containing the EARC system by maintaining the cells containing the EARC system in the absence of the inducer of the inducible activator-based gene expression system.

183. The method of claim 182, comprising:

185. 164. The method of claim 163, wherein the cells are removed after the treatment is completed.

186. 161. A cell according to claim 1 or a composition according to claim 160 for use in treating a disease or condition in a subject in need thereof.

187. 187. The cell of claim 186, wherein 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, enzyme or hormone deficiency, metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infectious disease, hemophilia, myocardial infarction, degenerative disease, age-related disease, or a disease or condition listed in Table 2.

188. 161. The cell of claim 1 or the composition of claim 160 for use in providing local immunosuppression at the site of transplantation in an allogeneic host.

189. 35. The cell of any one of claims 1 to 34, wherein one or more 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 that is in the top 5% of gene expression for all genes in the genome of the cell.

190. 35. The cell of any one of claims 1 to 34, 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 levels that are in the top 5% of gene expression for all genes in the genome of the cell.

191. 4. The cell of any one of claims 1 to 3, wherein one or more 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 that is in the top 5% of gene expression for all genes in the genome of the cell.

192. 4. The cell of any one of claims 1 to 3, 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 levels that are in the top 5% of gene expression for all genes in the genome of the cell.

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