Allograft tolerance without systemic immune containment

Abstract

The invention relates to allograft tolerance without systemic immune containment. There is provided a cell genetically modified to comprise at least one mechanism for providing localized immune containment at a transplantation site when transplanted into an allogeneic host; and methods for making and using the cells. The cell comprises a set of transgenes, each transgene encoding a gene product of cytoplasm, membrane binding or topical action, and having one or more of the following functions: mitigating antigen presenting cell activation and function; mitigating leukocyte activity or cytolytic function of the aggressive graft; the cytolytic function and phagocytosis of macrophages of allograft cells are reduced; inducing apoptosis in the graft-attacking leukocytes; reducing local inflammatory proteins; and protecting from leukocyte mediated apoptosis.

Description

[0001] This application is a divisional application. The filing date of the original application is June 12, 2018, the application number is 201880038734.5 (PCT / CA2018 / 050706), and the invention title is "Allograft Tolerance without Systemic Immune Suppression". Field of the Invention

[0002] The present disclosure generally relates to the field of transplantation. The present disclosure further relates to methods for generating local immune suppression in the context of transplanted cells. Background of the Invention

[0004] The emergence of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells has had a paradigm-shifting impact on regenerative and translational medicine. These cells can self-renew indefinitely in a pluripotent state while retaining the ability to differentiate into any cell type in the human body. Such properties have enabled researchers to better understand the causes of human development and developmental disorders. They have also provided modern medicine with powerful new tools for treating diseases that are difficult or impossible to treat with conventional medicine, including, for example, spinal cord injury, diabetes, blindness, multiple sclerosis, and cancer. As understanding of how to control stem cell differentiation and the biology of the differentiated cell products continues to grow, the efficacy and scope of cell therapies for applicable diseases will only increase.

[0005] These applications pose important and formidable challenges. Along with cell safety, one of the most important concerns is the immune rejection of cells from different genetic backgrounds. Immune rejection remains a key obstacle because the immune system has evolved a complex set of mechanisms to recognize and eliminate "non-self" cells that express specific protein fragments - especially those from the major histocompatibility complex (MHC in mice, HLA in humans) - that differ between donor and recipient (Yang et al., Nat Rev Genet. 18:309-26 (2017)). This response is almost certainly a byproduct of the evolutionary pressure to protect against opportunistic infections and malignancies, which are typically defined by the presence of "foreign" proteins and epitopes. Depending on the circumstances, rejection of the transplanted cells or tissue can occur on time scales of minutes / hours (hyperacute), days / months (acute), and months / years (chronic) (LaRosa et al., J Immunol. 178:7503-9 (2007)). This rejection is caused by the complex and coordinated effects of cell types from innate (Murphy et al., Immunol Rev. 241:39-48 (2011)) and adaptive immunity (Issa et al., Expert Rev Clin Immunol. 6:155-69 (2010)).

[0006] One of the most important pathways of 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 that are specific for the same peptides in secondary lymphoid organs (Lechler et al., J Exp Med. 155:31-41 (1982); Guermonprez et al., Annu Rev Immunol. 20:621-67 (2002); Stockwin et al., Immunol Cell Biol. 78:91-102 (2000)). These T cells then migrate to and kill the transplanted cells or tissues, with the release of cytolytic cytokines such as perforin and granzyme. NK cells can also induce apoptosis of donor cells based on foreign MHC expression or lack of MHC expression (Kitchens et al., Transplantation. 81:811-7 (2006); Benichou et al., Curr Opin OrganTransplant. 16:47-53 (2011)), and other cell types such as macrophages can support rejection by releasing pro-inflammatory cytokines at the site of implantation (Mannon, Curr Opin Organ Transplant. 17:20-5 (2012)). Many other cell types and subtypes also play a role in allograft rejection. Since these are the same immune pathways used to eliminate common viral and bacterial pathogens, they – along with allograft rejection – are highly conserved across vertebrate species.

[0007] Current solutions for preventing allograft rejection involve two options: finding a donor with a matching histocompatibility haplotype (most likely from a genetically related family), and much more commonly, using broadly directed immunosuppressive drugs (Wiseman, Clin J Am Soc Nephrol. 11:332-43 (2016); Malaise et al., Transplant Proc. 37:2840-2 (2005)). Common drugs include those from the following families: calcineurin inhibitors (Flechner et al., Clin Transplant. 22:1-15 (2008); Casey et al., Curr Opin Nephrol Hypertens. 20:610-5 (2011)), antiproliferatives (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 inhibit T cell proliferation or function (especially the first three). These drugs need to be taken daily for life, and missing even a single dose can increase the risk of rejection. However, they do not always work, and when they do, the chronic rejection rate still continues to climb over time (Demetris et al., Ann Transplant. 2:27-44 (1997); Libby et al., Immunity. 14:387-97 (2001)). Most importantly, they have systemic effects and ultimately leave the patient immunocompromised, with increased rates of cancer and life-threatening infections (Gallagher et al., J Am Soc Nephrol. 21:852-8 (2010)). In relation to ES cells, these drugs have shown only marginal improvement in allowing survival across the MHC barrier (Swijnenburg et al., Proc Natl Acad Sci USA. 105:12991-6 (2008); Toriumi et al., Neurol Res. 31:220-7 (2009)).Although newer and more targeted immunosuppressive agents have become available and been tested in the skin and heart (Larsen et al., Nature. 381:434-8 (1996)), as well as in the context of ES cell allografts (Pearl et al., Cell Stem Cell. 8:309-17 (2011)), they still act systemically and can thus compromise the host's immunity.

[0008] One proposed benefit of the discovery of iPS cells is that they can be generated from and for each patient. In theory, these cells should be protected from immune rejection by the corresponding patient (Pearl et al., Sci TranslMed. 4:164ps25 (2012)). However, inducing the iPS cell state involves epigenetic changes and in vitro culture stresses that can give rise to abnormalities and malignancies, and thus each cell line requires rigorous testing and / or genetic modification to achieve safety as well as 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 make this approach impractical both in practice and economically. Even if the cost were significantly reduced, it would not help those patients in need of immediate treatment for conditions such as burns, heart attacks, strokes, and spinal cord injuries, etc. In addition, given recent findings, there is still controversy over 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)).

[0009] In this regard, one proposed solution is to use natural inhibitory or regulatory immune cells, such as Tregs or others, which are expanded and / or transferred before, during, or after transplantation of therapeutic cells or tissues (Cobbold et al., Cold Spring Harb Perspect Med. 3(6)(2013); Wood et al., Nature reviews Immunology. 12:417-30(2012)). These strategies have been proposed based on the understanding of inhibitory immune pathways, particularly the discovery of the master regulator FoxP3 that 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 evidence of their crucial importance in promoting tolerance to allografts (Kendal et al., J Exp Med. 208:2043-53(2011)). This idea contrasts with some of the initial tolerance induction strategies that almost entirely focused on depleting effector T cells with monoclonal antibodies in combination with the generation of bone marrow grafts and donor chimerism (Cobbold et al., Nature. 323:164-6(1986); Qin et al., J Exp Med. 169:779-94(1989)). The importance of the inhibitory T cell phenotype was later realized, along with strategies that do not kill cells but block key T cell receptors in a way that renders them unresponsive to allografts (Cobbold et al., J Immunol. 172:6003-10(2004)) but are also able to inhibit naive T cells with other specificities (Cobbold et al., Immunol Rev. 129:165-201(1992); Qin et al., Eur J Immunol. 20:2737-45(1990)).These cells, now recognized as Tregs, can promote tolerance through multiple mechanisms, including (but not limited to) the expression of suppressive factors such as TGFβ (Nakamura et al., The Journal of experimental medicine. 194:629-44 (2001); Nakamura et al., J Immunol. 172:834-42 (2004)), CTLA4 (Tang et al., J Immunol. 181:1806-13 (2008); Walker et al., Trends Immunol. 36:63-70 (2015)), IL10 (O'Garra et al., J Clin Invest. 114:1372-8 (2004); Chaudhry et al., Immunity. 34:566-78 (2011)), and IL35 (Collison et al., Nature. 450:566-9 (2007)), as well as the 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)), and the depletion of 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 USA. 106:12055-60 (2009)).

[0010] Two approaches to the potential therapeutic use of Tregs involve the use of in vitro expansion of donor antigen together with transplantation, or the selective in vivo expansion that exploits the differences between regulatory T cells and effector T cells. Although these strategies are interesting, to date, long-term acceptance of allografts has not been demonstrated solely by the use of in vitro or in vivo expanded Tregs. There are still many complications and unknown aspects of Treg biology, including the optimal methods for in vitro or in vivo expansion, as well as treatment-related dosages and timing. It has also been shown that, depending on the inflammatory context, antigen-specific Treg suppression can be "overcome" (Korn et al., Nat Med. 13:423-31 (2007)), and Tregs can be killed by NK cells (Roy et al., J Immunol. 180:1729-36 (2008)).

[0011] In addition to Tregs, other suppressive cell types have been explored to induce allograft tolerance, such as antigen-presenting cells, like dendritic cells (DCs) (Walker et al., Trends Immunol. 36:63-70 (2015)). DCs are the link between innate and adaptive immunity, and they can induce effector and suppressive immune responses depending on circumstances such as their maturation state and local inflammatory cues. During allograft rejection, DCs present allograft antigens in the binding groove of MHC (mouse) or HLA (human) molecules on their surface together with co-stimulatory molecules such as CD80, CD86, and CD40, and allograft-specific T cell clones recognize these molecules and become activated (Walker et al., Trends Immunol. 36:63-70 (2015)). Tolerogenic DCs can be induced from an immature state by exposure to suppressive cues that keep the expression levels of MHC and co-stimulatory molecules low and that, in turn, promote naive T cells to become anergic or even T regulatory subtypes after DC-T cell interaction.

[0012] Therapeutically, one application of this biology is to expand DCs that are exposed in vitro to specific target allograft antigens and immune suppressive factors (many of which have been tested, including TGF-β, IL10, cAMP, prostaglandin E2, histamine, neuropeptides, vitamin D2, β2 agonists, HLA-G, glucosamine) as well as drugs such as corticosteroids, cyclosporine, tacrolimus, rapamycin, aspirin, mycophenolate mofetil, sanglifehrin, and deoxyspergualin (Hackstein et al., Nat Rev Immunol. 4:24-34 (2004)). Alternatively, DCs have been genetically engineered to directly express immunomodulatory factors such as TGF-β, IL-10, VEGF, FasL, CTLA4-Ig, IDO, NFKb decoy receptor, soluble TNFR, CCR7, and siRNA-induced IL-12 silencing (Morelli et al., Immunol Rev. 196:125-46 (2003)). These cultured or engineered DCs are then transferred together with the allograft into the recipient to test whether they can prolong allograft survival, assuming that they suppress allograft-specific T cells or increase the number of T regulatory cells in the allograft.

[0013] In one prototypical approach of this kind, bone marrow-derived DCs are transduced with SOCS1 (thereby preventing upregulation of costimulatory molecules and MHCII), which prolongs murine cardiac allografts (Fu et al., Cell Mol Immunol. 6:87-95 (2009)). In another demonstration, DCs expressing FasL are also able to prolong murine cardiac allografts (Min et al., J Immunol. 164:161-7 (2000)). Generally speaking, there are many single and combinatorial approaches using tolerogenic DCs according to these methods (Bjorck et al., J Heart Lung Transplant. 24:1118-20 (2005); Sun et al., PLoS One. 7:e52096 (2012); Li et al., J Immunol. 178:5480-7 (2007); Xu et al., Transplant Proc. 38:1561-3 (2006); Lan et al., J Immunol. 177:5868-77 (2006); Lutz et al., Eur J Immunol. 30:1813-22 (2000); Fischer et al., Transpl Immunol. 25:20-6 (2011)), and the results are highly variable depending on the type of 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 tests have begun to include safety testing in healthy volunteers (Dhodapkar et al., J Exp Med. 193:233-8 (2001); Dhodapkar et al., Blood. 100:174-7 (2002)) as well as a phase I clinical trial in 10 diabetic patients (Giannoukakis et al., Diabetes Care. 34:2026-32 (2011)).

[0014] There remain many unknowns regarding adoptive Treg and tolerogenic DC therapies, and one of the most important is the duration of their efficacy. Although in vivo studies have shown that prolonged allograft survival is possible using either of these two methods (with or without additional immunosuppressive drugs), it is not long-term, and in almost every case, the allograft ultimately dies. This is consistent with the fact that both Tregs and DCs have a limited time span. Additionally, especially in DCs, the tolerogenic phenotype has the potential to "convert" and instead promote inflammatory pathways (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 may be due to the highly adaptable nature of DCs and their ability to sense and respond to various inflammatory cues. These cells have also been shown to die extremely rapidly after adoptive transfer in vivo. There are also many subsets of suppressive Tregs and tolerogenic DCs that have been described, and it is currently unclear which is the ideal subtype, or whether it will depend entirely on the context of the allograft transplantation.

[0015] In addition, there are significant practical and economic barriers to these types of methods because they require clinicians to manipulate and handle complex immune cell types in addition to the treatment method. Given their limited lifespan, it remains unclear whether continuous and / or repeated delivery of these cells is required to confer long-term tolerance to allografts. This would complicate the already expensive and time-consuming methods used to culture, expand, or transduce cells with key immunomodulatory factors and ultimately hinder the use of treatments that are extremely time-sensitive.

[0016] Another approach for inducing tolerance is the use of hematopoietic cell transplantation (HCT), in which recipients of HLA-mismatched organs receive HCT with 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 can allow newly developed T and B cells in the recipient to tolerate 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 of hematopoietic cells in positive and negative selection in the thymus, where they eliminate cells with affinity for antigens contained in hematopoietic cells 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 the transplanted hematopoietic cells recognize recipient tissue as foreign and mount a systemic attack (Sun et al., PLoS One. 7:e52096 (2012)). From the outset, several HCT variants have been developed to suppress rejection, including the use of non-myeloablative strategies. These strategies use modified chemotherapy regimens, typically involving lower doses, such that recipients undergoing HCT do not receive complete ablation of their hematopoietic compartment. The latest of these strategies, for example, uses HCT based on facilitator cells (FC) that promote tolerance to facilitate tolerance in HLA-mismatched kidney recipients while largely avoiding GHVD (Leventhal et al., Sci Transl Med. 4:124ra28 (2012)).

[0017] In addition to the risks of GHVD and the risks of a secondary HCT procedure, a general limitation of these methods of inducing chimerism is the requirement that HLA-matched donors be available for harvesting bone marrow. While this is easily achieved in rodent studies, it is a rather demanding requirement in humans. Ideally, donor organs should be obtained from the donor as soon as possible, which would leave a very short window (if possible) for harvesting bone marrow from them. This is also an expensive and logistically demanding procedure that requires highly patient- and procedure-specific approaches. And, as with regulatory cell methods, it is not clear how to practically apply them to those situations where patients could benefit or require immediate treatment for acute injury or disease.

[0018] Another approach that has been tested for reducing allorejection in vivo is the removal of histocompatibility molecules (Torikai et al., Blood. 122:1341-9 (2013)), which are the major source of "non-self" recognition in allograft rejection. This is in line with empirical data showing that HLA-matched donors and recipients greatly improve organ survival rates after transplantation (Opelz et al., Rev Immunogenet. 1:334-42 (1999)). Although some positive results have been achieved using this approach, the removal of class I MHC renders cells highly vulnerable to NK cells (Pegram et al., Immunol Cell Biol. 89:216-24 (2011); Raulet et al., Nat Rev Immunol. 6:520-31 (2006); Huntington, Immunol Cell Biol. 92:208-9 (2014)). It also leaves intact non-MHC-dependent killing pathways among CD8+ T cells (Haspot et al., Am J Transplant. 14:49-58 (2014)), and does not address antigenic differences outside the MHC / HLA gene family (minor antigens) (Roopenian et al., Immunol Rev. 190:86-94 (2002)).

[0019] The adoption of this method involves the deletion of all classical HLA class I molecules from pluripotent stem cells, in combination with the introduction of a gene encoding HLA-E, a minimally polymorphic HLA that inhibits NK cells (Gornalusse et al., Nat Biotechnol. (2017)). Although this method has shown short-term resistance to NK and CD8 T cell attack in some immunocompromised humanized mice, it has not been demonstrated that these cells can 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 was accompanied by a severe limitation in that CTLA4-Ig can lead to systemic immune suppression. It has not been demonstrated that a set of modifications to ES or iPS cells allows them to escape allograft rejection without the possibility of systemic immune suppression and without the need for immunosuppressive drugs.

[0020] The object of the present disclosure is to mitigate and / or eliminate one or more of the above deficiencies. Summary of the Invention

[0021] In one aspect, there is provided a cell that has been genetically modified to include at least one mechanism for providing local immune suppression at the site of transplantation when transplanted into an allogeneic host. The genetically modified cell includes: a set of transgenes, each transgene encoding a cytoplasmic, membrane-bound, or locally acting gene product; and having one or more of the following functions: a) reducing antigen-presenting cell activation and function; b) reducing the activity or lytic function of white blood cells that attack the graft; c) reducing the macrophage lytic function and phagocytosis of allogeneic graft cells; d) inducing apoptosis of white blood cells that attack the graft; e) reducing local inflammatory proteins; and f) protecting against white blood cell-mediated apoptosis.

[0022] In one embodiment of the cell, the set of transgenes includes one or more of the following genes (e.g., one, two, three, four, five, six, seven, or all eight): PD-L1, HLA-G (or the murine form of HLA-G, H2-M3), Cd47, Cd200, FASLG (or the murine form of FASLG, FasL), Ccl21 (or the murine form of Ccl21, Ccl21b), Mfge8, and Serpin B9 (or the murine form of Serpin B9, Spi6).

[0023] In one embodiment of the cell, the set of transgenes includes two or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0024] In one embodiment of the cell, the set of transgenic genes includes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6), or genes encoding biologics that act as agonists of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0025] In one embodiment of the cell, the cell further comprises one or more of the following transgenes (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or all eleven): TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or genes encoding biologics that act as agonists of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.

[0026] In one embodiment of the cell, the TGF-β or biologic acts locally in the graft environment. In one embodiment of the cell, the TGF-β or biologic acts locally with minimal systemic effects in the graft environment.

[0027] In various embodiments of the cells, the cells are a source of stem cells, cells suitable for genome editing, and / or therapeutic cell types (e.g., cells that can differentiate into therapeutic cell types or cells of a desired target tissue). In various embodiments, the cells are 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 progenitors, germline stem cells, lung stem cells or progenitors, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitors, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitors, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte precursors, B cells, exocrine cells, pancreatic progenitors, endocrine progenitors, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroleukemia cell line, osteocytes, synoviocytes, tendon cells, ligament cells, meniscus cells, adipocytes, dendritic cells, natural killer cells, skeletal muscle cells, cardiomyocytes, erythroid-megakaryocytes, eosinophils, macrophages, T cells, pancreatic islet beta cells, neurons, cardiomyocytes, blood cells, exocrine progenitors, duct cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, cholangiocytes, white or brown adipocytes, hormone-secreting cells, epidermal keratinocytes, epithelial cells, renal cells, skeletal joint synoviocytes, periosteal cells, perichondrial cells, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amnion or placental membranes, serosal cells, somatic cells, or cells derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach.

[0028] In one embodiment of the cell, the cell is further genetically modified to include at least one (e.g., one, two, three, or more) mechanisms for controlling cell proliferation (e.g., to reduce the oncogenic potential of the modified cell or to reduce the proliferation of modified cells that have become oncogenic). The genetically modified cell includes: genetic modification of one or more (e.g., one, two, three, or more) cell division loci / locus (CDL), where the CDL is one or more loci whose transcript is expressed by dividing cells (e.g., all dividing cells contain one or more of the immune containment transgenes), and the genetic modification is one or more of the following: a) an abolish link (ALINK) system, which includes a DNA sequence encoding a negative selectable marker transcriptionally linked to the DNA sequence encoding the CDL; and b) a CDL-regulated exogenous activator (EARC) system, which includes an inducible activator-based gene expression system operably linked to the CDL.

[0029] In one embodiment of the cell, the genetic modification of the CDL includes targeted replacement of the CDL with one or more of the following: a) a DNA vector containing the ALINK system; b) a DNA vector containing the EARC system; c) a DNA vector containing the ALINK system and the EARC system; where the ALINK and / or EARC system is each operably linked to the CDL.

[0030] 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 modification can only be produced by the EARC-modified allele.

[0031] In various embodiments of the cell, the CDL is one or more (e.g., one, two, three, or more) of the loci listed in Table 5. In various embodiments, the CDL encodes a gene product that functions in one or more of the following: cell cycle, DNA replication, RNA transcription, protein translation, and metabolism. In various embodiments, the CDL is one or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5, and Eef2 / EEF2, preferably the CDL is Cdk1 or CDK1. In some embodiments, the CDL is Top2A. In some embodiments, the CDL is Eef2. In various embodiments, the CDL is two or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5, and Eef2 / EEF2, preferably the CDL is Cdk1 and Top2A or Cdk1 and Eef2.

[0032] In various embodiments of the cell, the ALINK system includes the herpes simplex virus-thymidine kinase / ganciclovir system, the cytosine deaminase / 5-fluorocytosine system, the carboxylesterase / irinotecan system, or the iCasp9 / AP1903 system, preferably the ALINK system is the herpes simplex virus-thymidine kinase / ganciclovir system.

[0033] In various embodiments of the cell, the EARC system is the doxycycline-inducible "dox-bridge" system, the coumarate-switch-inducible system, the ecdysone-inducible system, the radio wave-inducible system, or the ligand-reversible dimerization system, preferably the EARC system is the dox-bridge system.

[0034] In one aspect, a method for providing local immune containment at a transplantation site in an allogeneic host is provided. The method includes providing cells; and expressing a set of transgenes in the cells, each transgene encoding a gene product that is cytoplasmic, membrane-bound, or locally acting; and having one or more of the following functions: a) reducing antigen-presenting cell activation and function; b) reducing the activity or lytic function of white blood cells that attack the graft; c) reducing the macrophage lytic function and phagocytosis of allogeneic graft cells; d) inducing apoptosis of white blood cells that attack the graft; e) reducing local inflammatory proteins; and f) protecting against white blood cell-mediated apoptosis.

[0035] In one embodiment of the method, the group of transgenes comprises one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6), or a gene encoding a 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).

[0036] In one embodiment of the method, the group of transgenes comprises two or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0037] In one embodiment of the method, the group of transgenes comprises PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6), or a gene encoding a biological agent that acts as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0038] In one embodiment of the method, the method further comprises expressing one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or all eleven) of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or a gene encoding a biological agent that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1d39. In one embodiment, the TGF-β or biological agent acts locally in the graft environment. In one embodiment of the cell, the TGF-β or biological agent acts locally in the graft environment with minimal systemic effects.

[0039] In various embodiments of the method, the cells are a source of stem cells, cells suitable for genome editing, and / or therapeutic cell types (e.g., cells that can differentiate into therapeutic cell types or cells of a desired target tissue). In various embodiments, the cells are 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 progenitors, germline stem cells, lung stem cells or progenitors, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitors, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitors, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte precursors, B cells, exocrine cells, pancreatic progenitors, endocrine progenitors, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroleukemia cell line, osteocytes, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, natural killer cells, skeletal muscle cells, cardiomyocytes, erythroid-megakaryocytes, eosinophils, macrophages, T cells, pancreatic islet beta cells, neurons, cardiomyocytes, blood cells, exocrine progenitors, duct cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, cholangiocytes, white or brown adipocytes, hormone-secreting cells, epidermal keratinocytes, epithelial cells, renal cells, skeletal joint synoviocytes, periosteal cells, perichondrial cells, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amniotic or placental membranes, serosal cells, somatic cells, or cells derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.

[0040] In various embodiments of the method, the cells are provided (e.g., injected) to or near the transplantation site. In various embodiments of the method, the cells are provided (e.g., injected or implanted) into a graft (e.g., injected or implanted into a tissue or organ graft before, during, or after transplantation). In some embodiments, the cells expressing the transgene are cells of the graft (e.g., cells of the transplanted tissue or organ are modified to express one or more of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., one, two, three, four, five, six, seven, or all eight)).

[0041] In one aspect, a method of controlling cell proliferation at a transplantation site in an allogeneic host is provided (e.g., reducing the oncogenic potential of cells at the transplantation site or reducing the proliferation of cells that have become oncogenic at the transplantation site). The method comprises: a) genetically modifying one or more (e.g., one, two, three or more) cell division loci / genes (CDLs) in the cells, the CDLs being one or more loci whose transcripts are expressed by dividing cells (e.g., all dividing cells contain one or more of immunosuppressive transgenes), the genetic modification of the CDLs comprising one or more of the following: i) an ablation-link (ALINK) system, the ALINK system comprising a DNA sequence encoding a negative selectable marker transcriptionally linked to the DNA sequence encoding the CDL; and i) a CDL-regulated inducible exogenous activator (EARC) system, the EARC system comprising an inducible activator-based gene expression system operably linked to the CDL; b) genetically modifying the cells to comprise at least one mechanism for providing local immunosuppression at the transplantation site; c) transplanting the cells or the cell population at a transplantation site in an allogeneic host; and d) allowing proliferation of the genetically modified cells comprising the ALINK system by maintaining the genetically modified cells comprising the ALINK system in the absence of an inducer of the negative selectable marker, and / or eliminating and / or inhibiting proliferation of the cells comprising the ALINK system by exposing the genetically modified cells comprising the ALINK system to an inducer of the negative selectable marker; and / or allowing proliferation of the genetically modified cells comprising the EARC system by exposing the genetically modified cells comprising the EARC system to an inducer of the inducible activator-based gene expression system, or preventing or inhibiting proliferation of the cells comprising the EARC system by maintaining the genetically modified cells comprising the EARC system in the absence of an inducer of the inducible activator-based gene expression system.

[0042] In one embodiment of the method, the genetic modification of the CDL comprises targeted replacement of the CDL with one or more of the following: a) a DNA vector comprising the ALINK system; b) a DNA vector comprising the EARC system; c) a DNA vector comprising the ALINK system and the EARC system; wherein the ALINK and / or EARC system is each operably linked to the CDL.

[0043] 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 modification can only be produced by the allele modified by ERAC.

[0044] 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 following: cell cycle, DNA replication, RNA transcription, protein translation and metabolism. In various embodiments, the CDL is one or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5 and Eef2 / EEF2, preferably the CDL is Cdk1 or CDK1. In some embodiments, the CDL is Top2A. In some embodiments, the CDL is Eef2. In various embodiments, the CDL is two or more of Cdk1 / CDK1, Top2A / TOP2A, Cenpa / CENPA, Birc5 / BIRC5 and Eef2 / EEF2, preferably the CDL is Cdk1 and Top2A or Cdk1 and Eef2.

[0045] In various embodiments of the method, the ALINK system includes 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.

[0046] In various embodiments of the method, the EARC system is a doxycycline-inducible "dox-bridge" system, a coumarate-switch-inducible system, an ecdysone-inducible system, a radio wave-inducible system or a ligand-reversible dimerization system, preferably the EARC system is a dox-bridge system.

[0047] In one embodiment of the method, the genetically modified cell comprises: a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane-bound or locally acting; and having one or more of the following functions: a) reducing antigen-presenting cell activation and function; b) reducing the activity or lytic function of white blood cells that attack the graft; c) reducing the macrophage lytic function and phagocytosis of allogeneic graft cells; d) inducing apoptosis of white blood cells that attack the graft; e) reducing local inflammatory proteins; and f) protecting against white blood cell-mediated apoptosis.

[0048] In one embodiment of the method, the group of transgenes comprises one or more (e.g., one, two, three, four, five, six, seven, or all eight) of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6), or a gene encoding a 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).

[0049] In one embodiment of the method, the group of transgenes comprises two or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0050] In one embodiment of the method, the group of transgenes comprises PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6), or a gene encoding a biological agent that acts as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0051] In one embodiment of the method, the cell further comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or all eleven) of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39, or a gene encoding a biological agent that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39.

[0052] In one embodiment of the method, the TGF-β or the biological agent acts locally in the graft environment. In one embodiment, the TGF-β or the biological agent acts locally with minimal systemic effects in the graft environment.

[0053] In various embodiments of the method, the cells are a source of stem cells, cells suitable for genome editing, and / or therapeutic cell types (e.g., cells that can differentiate into therapeutic cell types or cells of a desired target tissue). In various embodiments, the cells are 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 progenitors, germline stem cells, lung stem cells or progenitors, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitors, multipotent stem cells, amniotic stem cells, umbilical cord blood stem cells, neural stem cells or progenitors, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, monocyte precursors, B cells, exocrine cells, pancreatic progenitors, endocrine progenitors, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroleukemia cell line, osteocytes, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, natural killer cells, skeletal muscle cells, cardiomyocytes, erythroid-megakaryocytes, eosinophils, macrophages, T cells, pancreatic islet beta cells, neurons, cardiomyocytes, blood cells, exocrine progenitors, duct cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, cholangiocytes, white or brown adipocytes, hormone-secreting cells, epidermal keratinocytes, epithelial cells, renal cells, skeletal joint synoviocytes, periosteal cells, perichondrial cells, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amniotic or placental membranes, serosal cells, somatic cells, or cells derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach.

[0054] In various embodiments of the method, the cells are provided (e.g., injected) to or near the transplantation site. In various embodiments of the method, the cells are provided (e.g., injected or implanted) into a graft (e.g., injected or implanted into a tissue or organ graft before, during, or after transplantation). In some embodiments, the cells expressing the transgene are cells of the graft (e.g., cells of the transplanted tissue or organ are modified to express one or more of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., one, two, three, four, five, six, seven, or all eight)).

[0055] 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.

[0056] In various embodiments of the method, the host has a degenerative disease or disorder treatable by cell therapy. In various embodiments, the disease or disorder is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, nervous system disease (such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS), enzyme or hormone deficiency, metabolic disorder (e.g., lysosomal storage 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 hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infectious disease, hemophilia, degenerative disease (e.g., Charcot-Marie-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, keratoglobus, muscular dystrophy, neuronal ceroid lipofuscinosis, pre-existing disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age-related disease (e.g., atherosclerosis, cardiovascular disease (e.g., angina, myocardial infarction), cataract, osteoporosis, or hypertension).

[0057] In some embodiments of any of the foregoing aspects, one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level equal to or greater than the expression level of the corresponding endogenous gene in activated white blood cells (e.g., T cells, e.g., the expression level of the transgene to be masked is equal to the expression level of the endogenous gene in activated white blood cells, or 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more higher than the expression level of the endogenous gene in activated white blood cells). 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 a level equal to or greater than the expression level of the corresponding endogenous gene in activated white blood cells.

[0058] In some embodiments of any of the foregoing aspects, one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level greater than the expression level of the corresponding endogenous gene in wild-type stem cells (e.g., wild-type ES cells from the same species, e.g., the expression level of the shielded transgene is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1,000-fold or higher in the shielded cells compared to the expression of the endogenous gene in unmodified wild-type ES cells from the same species). In some embodiments, all 8 of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level greater than the expression level of the endogenous gene (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100-fold or higher) in wild-type stem cells (e.g., embryonic stem cells from the same species as the shielded cells). In some embodiments, one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level within the top 5% of the gene expression of all genes in the ES cell genome. In some embodiments, one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level within the top 1% of the gene expression of all genes in the ES cell genome. In some embodiments, all of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level within the top 5% of the gene expression of all genes in the ES cell genome.

[0059] In some embodiments of any of the foregoing aspects, the PD-L1 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0060] 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% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 15.

[0061] 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% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0062] 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% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

[0063] 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% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 9.

[0064] 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% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1.

[0065] 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% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0066] 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% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 7.

[0067] 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% sequence identity or higher identity) to the amino acid sequence of SEQ ID NO: 17.

[0068] In some embodiments of any of the foregoing aspects, the one or more transgenes are operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is selected from the group consisting of: CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter.

[0069] In some embodiments of any of the foregoing aspects, the cell further comprises (e.g., the cell is further modified to comprise) a transgene encoding a therapeutic agent. In some embodiments, the therapeutic agent is a protein or an antibody. In some embodiments, the antibody is an inhibitory antibody or an agonist antibody. In some embodiments, the therapeutic agent is an agent listed in Table 2. In some embodiments, the therapeutic agent is the wild-type form of a gene mutated in a subject (e.g., the wild-type form of a gene mutated in the subject's disease or disorder, e.g., a genetic mutation associated with cancer, enzyme or hormone deficiency, metabolic disorder or degenerative disease). In some embodiments, an inducible expression system selected from the group consisting of a tetracycline response element, a light-inducible system, a radiogenetic system, a coumarate switch-inducible system, an ecdysone-inducible system, a destabilizing domain system or a ligand-reversible dimerization system is used to express the therapeutic agent. In some embodiments, 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, a tk promoter of HSV, a mouse mammary tumor virus (MMTV) promoter, an LTR promoter of HIV, a promoter of Moloney virus, an Epstein-Barr virus (EBV) promoter and a Rous sarcoma virus (RSV) promoter is used to express the therapeutic agent.

[0070] In another aspect, there is provided a population of genetically modified cells according to any of the above cells.

[0071] In one aspect, there is provided a method for providing local immune containment at a transplantation site in an allogeneic host. The method comprises transplanting the genetically modified cell or the population of genetically modified cells as described above at the transplantation site in an allogeneic host.

[0072] In another aspect, the invention features a composition comprising the cells of the invention. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0073] In another aspect, there is provided a kit comprising the cells of the invention or the pharmaceutical composition of the invention.

[0074] In another aspect, there is provided a method of treating a disease or disorder 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 disorder is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, 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, Graves' disease, inflammatory bowel disease, Addison's disease, Sjogren's syndrome), Hashimoto's thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infectious disease, hemophilia, 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, keratoglobus, muscular dystrophy, neuronal ceroid lipofuscinosis, pre-existing disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age-related disease (e.g., atherosclerosis, cardiovascular disease (e.g., angina, myocardial infarction), cataract, osteoporosis, or hypertension) or a disease or disorder listed in Table 2, and / or the cell further comprises a transgene encoding the corresponding therapeutic agent listed in Table 2 or the wild-type form of a gene mutated in the subject (e.g., the wild-type form of a gene mutated in relation to the disease or disorder of the subject, such as a genetic mutation associated with cancer, enzyme or hormone deficiency, metabolic disorder, or degenerative disease). In some embodiments, the disease or disorder 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 VEGF receptor (e.g., soluble VEGFR-1 or NRP-1), platelet factor-4, prolactin, SPARC, a VEGF inhibitory antibody (e.g., bevacizumab or ranibizumab) or a soluble decoy receptor described in Holash et al., Proc Natl Acad Sci U.S.A. 99:11383-11398, 2002, e.g., VEGF-Trap 亲本 VEGF-Trap ΔB1 VEGF-TrapΔB2 , VEGF-Trap R1R2 , such as aflibercept). In some embodiments, the disease or disorder is osteoarthritis or rheumatoid arthritis, and the therapeutic agent is an anti-inflammatory biologic (such as a TNFα inhibitor (e.g., adalimumab, etanercept, infliximab, golimumab, or certolizumab), an interleukin-6 (IL6) receptor inhibitor (e.g., tocilizumab), an IL1 receptor inhibitor (e.g., anakinra), or another agent for treating rheumatoid arthritis (e.g., abatacept, rituximab)). In some embodiments, the disease or disorder is diabetes (e.g., type 1 diabetes or type 2 diabetes), and the therapeutic agent is insulin. In some embodiments, the disease or disorder is hemophilia, and the therapeutic agent is factor VIII. In some embodiments, the disease or disorder is a metabolic defect, and the therapeutic agent is a transgenic nucleic acid sequence having the wild-type form of a gene that is mutated in the subject or a transgenic that encodes an enzyme that is lacking in the subject.

[0075] In some embodiments of any of the foregoing aspects, prior to administration to a subject, the cells are differentiated into a lineage-restricted cell type. In some embodiments, the disease or disorder is myocardial infarction, and the cells are differentiated into cardiomyocytes. In some embodiments, the disease or disorder is blindness, and the cells are differentiated into photoreceptor cells. In some embodiments, the disease or disorder is spinal cord injury, Parkinson's disease, Huntington's disease, or Alzheimer's disease, and the cells are differentiated into neurons. In some embodiments, the disease or disorder is multiple sclerosis, and the cells are differentiated into glial cells.

[0076] In some embodiments of any of the foregoing aspects, the cells are administered locally (e.g., by injection or implantation) to a tissue or body part in need of the cells or the therapeutic agent.

[0077] In some embodiments of any of the foregoing aspects, the cells are administered intravenously, subcutaneously, intramuscularly, transdermally, intradermally, parenterally, intraarterially, intravascularly, or by perfusion.

[0078] In some embodiments of any of the foregoing aspects, the cells are administered by subcutaneous injection to create a subcutaneous tissue mass.

[0079] In some embodiments of any of the foregoing aspects, the cells are administered as a tissue. In some embodiments, the tissue is administered together with a gel, a biocompatible matrix, or a cell scaffold.

[0080] In some embodiments of any of the foregoing aspects, the cells are administered in an amount of 25,000 to 5,000,000,000 cells (e.g., 2.5x10 4 , 5x10 4 , 7.5x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 6x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 or 5x10 9 cells).

[0081] In some embodiments of any of the foregoing aspects, the cells are administered in an amount of 800,000,000 to 100,000,000,000 cells (e.g., 8x10 8 , 9x10 8 , 1x10 9 , 2x109 , or 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , 9x10 10 or 1x10 11 administered in an amount of (number of cells).

[0082] In some embodiments of any of the foregoing methods, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered before administering the cells. In some embodiments, the additional therapeutic agent is administered after administering the cells. In some embodiments, the additional therapeutic agent is administered concurrently with administering the cells.In some embodiments, the additional therapeutic agent is an immunosuppressive agent, a disease-modifying anti-rheumatic drug (DMARD), a biological response modifier (a type of DMARD), a corticosteroid or non-steroidal anti-inflammatory drug (NSAID), prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, tofacitinib, adalimumab, abatacept, anakinra, anakinra (kineret), 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, cyclosporin, dimethyl fumarate, etanercept, fingolimod, fumarates, colchicine, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonists, long-acting beta2 agonists, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, probiotics, retinoids, salicylic acid, short-acting beta2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab or vedolizumab, bevacizumab, ranibizumab or aflibercept), photodynamic therapy, photocoagulation, carbidopa-levodopa, dopamine agonists, MAO-B inhibitors, catechol-O-methyltransferase inhibitors, anticholinergics, amantadine, deep brain stimulation, anticoagulants, antiplatelet agents, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, angiotensin receptor neprilysin inhibitors, beta blockers, combined alpha and beta blockers, calcium channel blockers, cholesterol-lowering drugs, niacin, cholesterol absorption inhibitors, digitalis preparations, diuretics, vasodilators, dual antiplatelet therapy, cardiac surgery, antiviral compounds, nucleoside analogue 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, alpha-glucosidase inhibitors, bile acid sequestrants, aspirin, dietary regimens, clotting factors, desmopressin, clot-preserving drugs, fibrin sealants, physical therapy, coenzymes, bone marrow grafts, organ grafts, hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium-chain triglycerides, miglustat, enzyme replacement therapy, checkpoint inhibitors, chemotherapeutic drugs, biologic drugs, radiotherapy, cryotherapy, hyperthermia, surgical removal of tumor tissue or anti-cancer vaccines.

[0083] In some embodiments of any of the foregoing methods, the method further comprises controlling the proliferation of cells. In some embodiments, the cells comprise an ALINK system, and the method of controlling proliferation comprises: i) allowing the proliferation of cells comprising the ALINK system by maintaining the cells comprising the ALINK system in the absence of an inducer of a negative selectable marker; or ii) eliminating or inhibiting the proliferation of cells comprising the ALINK system by exposing the cells comprising the ALINK system to an inducer of a negative selectable marker. In some embodiments, the cells comprise an EARC system, and the method of controlling cell proliferation comprises: i) allowing the proliferation of cells comprising the EARC system by exposing the cells comprising the EARC system to an inducer of an inducible activator-based gene expression system; or ii) preventing or inhibiting the proliferation of cells comprising the EARC system by maintaining the cells comprising the EARC system in the absence of an inducer of an inducible activator-based gene expression system.

[0084] In some embodiments of any of the foregoing methods, the cells are removed after completion of treatment. Removal of the cells can be by surgery (e.g., removal of a transplanted tissue or organ, or removal of subcutaneous tissue that is obscured), or by using the ALINK and / or EARC system. In some embodiments, one or more (e.g., one, two, three, four, or more) ALINK and / or EARC systems are used to eliminate all obscured cells.

[0085] In another aspect, the present invention provides the cells of the present invention or the compositions of the present invention for treating a disease or disorder in a subject in need thereof. In some embodiments, the disease or disorder is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, neurological diseases (such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS), enzyme or hormone deficiency, metabolic disorders (e.g., lysosomal storage disorders, galactosemia, maple syrup urine disease, phenylketonuria, glycogen storage disease, mitochondrial disorders, Friedrich's ataxia, peroxisomal disorders, metal metabolism disorders, or organic acidemias), autoimmune diseases (e.g., psoriasis, systemic lupus erythematosus, Graves' disease, inflammatory bowel disease, Addison's disease, Sjogren's syndrome), Hashimoto's thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infectious diseases, hemophilia, degenerative diseases (e.g., Charcot-Marie-Tooth disease, 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, keratoglobus, muscular dystrophy, neuronal ceroid lipofuscinosis, pre-existing disease, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age-related diseases (e.g., atherosclerosis, cardiovascular diseases (e.g., angina, myocardial infarction), cataract, osteoporosis, or hypertension), or a disease or disorder listed in Table 2.

[0086] In another aspect, the present invention provides the cells of the present invention or the compositions of the present invention for providing local immune containment at the site of transplantation in an allogeneic host.

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

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

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

[0090] In some embodiments of any of the foregoing aspects, the cell comprises five of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (for example, PD-L1, HLA-G (H2-M3), Cd47, Cd200, and FASLG (FasL); PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Mfge8, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), and Mfge8, PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), and 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 SerpinB9 (Spi6)). In some embodiments of any of the foregoing aspects, the cell comprises six of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, Mfge8, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), Ccl21 (Ccl21b), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), Mfge8, and SerpinB9 (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)).;

[0091] In some embodiments of any of the foregoing aspects, the cell comprises seven of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Mfge8, and Serpin 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)).

[0092] In some embodiments of any of the foregoing aspects, the cell comprises all eight of the following group of transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

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

[0094] In some embodiments of any of the foregoing aspects, the cell comprises one or more (e.g., one, two, three, four, five, six, or all seven) of the following transgenes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0095] In some embodiments of any of the foregoing aspects, the cell comprises one or more (e.g., one, two, three, four, five, or all six) of the following transgenes: HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0096] In some embodiments of any of the foregoing aspects, the cell is not modified to express PD-L1.

[0097] In some embodiments of any of the foregoing aspects, the cell is not modified to express FasL.

[0098] In some embodiments of any of the foregoing aspects, the cell is not modified to express TGF-β. In some embodiments of any of the foregoing aspects, the cell is not modified to express CTLA4 or CLTA4-Ig. In some embodiments of any of the foregoing aspects, the cell is not modified to express IDO. In some embodiments of any of the foregoing aspects, the cell is not modified to express IL-35. In some embodiments of any of the foregoing aspects, the cell is not modified to express IL-10. In some embodiments of any of the foregoing aspects, the cell is not modified to express VEGF. In some embodiments of any of the foregoing aspects, the cell is not modified to express NFkb decoy receptor. In some embodiments of any of the foregoing aspects, the cell is not modified to express soluble TNFR. In some embodiments of any of the foregoing aspects, the cell is not modified to express CCR7. In some embodiments of any of the foregoing aspects, the cell is not modified to express SOCS1. In some embodiments of any of the foregoing aspects, the cell is not modified to express HLA-E. In some embodiments of any of the foregoing aspects, the cell is not modified to express siRNA against IL-12.

[0099] Definitions

[0100] Unless otherwise defined, 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.

[0101] As used herein, the term "about" refers to a value that is no more than 10% higher or lower than the described value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0102] As used herein, the term "activated leukocyte" refers to the state of a leukocyte (e.g., granulocyte, such as neutrophil, eosinophil, or basophil; monocyte or lymphocyte, such as B or T cell) that is caused by a response to a perceived injury. When leukocytes become activated, they can proliferate, secrete cytokines, differentiate, present antigens, become more polarized, become more phagocytic, and / or become more cytotoxic. Factors that stimulate immune cell activation include pro-inflammatory 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 employed by those skilled in the art (see, e.g., Frauwith and Thompson, J. Clin. Invest 109:295-299 (2002); and Trickett and Kwan, J. Immunol. Methods 275:251-255 (2003)).

[0103] As used herein, "allogeneic" refers to cells, tissues, DNA, or factors taken from or derived from different subjects of the same species.

[0104] As used herein, the term "stem cell" refers to a cell that can differentiate into one or more specialized cells and has the ability to self-renew. Stem cells include pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); and multipotent stem cells, such as umbilical cord blood stem cells, mesenchymal stromal cells, and adult stem cells, which are found in various tissues. The term "stem cell" also includes cells suitable for genome editing, cells that can serve as a source of therapeutic cell types (e.g., cells that can be directed to differentiate into lineage-restricted or terminally differentiated cells for cell therapy, or cells of the desired target tissue), and cells with "artificial" stem cell properties obtained (e.g., pluripotency or multipotency or self-renewal).

[0105] As used herein, the terms "embryonic stem cell" and "ES cell" refer to totipotent or pluripotent stem cells of embryonic origin derived from the inner cell mass of a blastocyst that can be maintained in vitro culture under suitable conditions. ES cells are capable of differentiating into cells of any of the three vertebrate germ layers (e.g., endoderm, ectoderm, or mesoderm). ES cells are also characterized by their ability to proliferate indefinitely under suitable in vitro culture conditions. See, e.g., Thomson et al., Science 282:1145 (1998).

[0106] As used herein, the terms "induced pluripotent stem cell", "iPS cell", and "iPSC" refer to pluripotent stem cells that can be directly derived from differentiated somatic cells. Human iPS cells can be generated by introducing a specific set of reprogramming factors into non-pluripotent cells, which can include, for example, Oct3 / 4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Soxl5), Myc family transcription factors (e.g., c-Myc, 1-Myc, n-Myc), Kruppel-like family (KLF) transcription factors (e.g., KLF1, KLF2, KLF4, KLF5), and / or related transcription factors such as NANOG, LIN28, and / or Glis1. Human iPS cells can also be generated, for example, by using miRNAs, small molecules that mimic the action of transcription factors, or lineage specifiers. Human iPS cells are characterized by their ability to differentiate into any cell of the three vertebrate germ layers (e.g., endoderm, ectoderm, or mesoderm). Human iPS cells are also characterized by their ability to proliferate indefinitely under suitable in vitro culture conditions. See, e.g., Takahashi and Yamanaka, Cell 126:663 (2006).

[0107] As used herein, the term "attenuating antigen presenting cell activation and function" refers to a transgene encoding a gene product that functions to inhibit antigen presenting cell activation or the ability of antigen presenting cells to promote the activation of white blood cells that attack a graft (Fiorentino et al., J Immunol. 146:3444-51 (1991); Salio et al., Eur J Immunol. 29:3245-53 (1999)). In one embodiment, attenuation of antigen presenting cell activation and function refers to a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of APC activation and function relative to a control (e.g., as determined using an assay for antigen presenting cell activation such as a reduction in proliferation, a reduction in 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 media or patient samples such as blood samples), or a reduction in the levels of cell surface markers (e.g., CD11c, CD11b, HLA molecules (e.g., MHC-II), CD40, B7, IL-2, CD80 or CD86, which can be evaluated using flow cytometry, immunohistochemistry, in situ hybridization and other assays that permit 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 antigen. 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 "masking" or "masked" genes.

[0108] As used herein, the term "attenuating leukocyte activity or lytic function against a graft" refers to a transgene encoding a gene product that functions to inhibit or prevent leukocyte activity or lytic function against a graft in the vicinity of allogeneic transplanted cells (MacDonald et al., J Immunol. 126:1671-5 (1981); Bongrand et al., Eur J Immunol. 13:424-9 (1983); MacDonald et al., Eur J Immunol. 9:466-70 (1979)). In one embodiment, attenuation of leukocyte activity or lytic function against a graft refers to a reduction in leukocyte activity or lytic function by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control (e.g., as determined using an assay for leukocyte activation, such as a reduction in proliferation, or a reduction in 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 γ (IFNγ) and granulocyte macrophage colony-stimulating factor (GMCSF), which can be measured using ELISA or western blot analysis of a culture medium or patient sample (such as a blood sample)), or a reduction in polarization (e.g., a reduction in the levels of IL-12, TNF, IL-1β, IL-6, IL-23, MARCO, MHC-II, CD86, iNOS, CXCL9 and CXCL10 in macrophages or monocytes, or a reduction in the levels of Th1-specific markers (e.g., T-bet, IL-12R, STAT4), chemokine receptors (e.g., CCR5, CXCR6 or CXCR3) or Th2-specific markers: (e.g., CCR3, CXCR4, STAT6, GATA3 or IL-4Rα) in T cells, which can be evaluated using flow cytometry, immunohistochemistry, in situ hybridization, qPCR or western blot analysis for cell markers or intracellular proteins and ELISA or protein analysis for secreted proteins); or as determined using an assay for lytic function (e.g., by incubating leukocytes 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 a fluorescent viability stain or by measuring the secretion of lytic granules (e.g., perforin, granzyme or other lytic proteins released from immune cells) from leukocytes). Methods for determining attenuation of leukocyte activity or lytic function against a graft are known in the art. Examples of gene products that attenuate leukocyte activity or lytic function against a graft include, but are not limited to: PD-L1, HLA-G (H2-M3), Cd39, Cd73 and Lag3.Such transgenes may be referred to herein as "masking" or "masked" genes.

[0109] As used herein, the term "attenuating macrophage cytolytic function and phagocytosis of allogeneic graft cells" refers to a transgene encoding a gene product whose function is to inhibit or prevent macrophage cytolytic function and / or phagocytosis of allogeneic graft cells (Fish et al., Toxicology. 19:127-38. (1981); Sung et al., J Biol Chem. 260:546-54 (1985); Amash et al., J Immunol. 196:3331-40 (2016)). In one embodiment, attenuation of macrophage cytolytic function and phagocytosis of allogeneic graft cells refers to a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% in macrophage cytolytic function and / or phagocytosis of allogeneic graft 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 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 a fluorescent viability stain or by measuring the secretion of cytolytic granules (e.g., perforin, granzyme or other cytolytic proteins released from immune cells) from the macrophages; or as determined using an assay for macrophage phagocytosis (e.g., culturing macrophages with fluorescent beads or a target cell line that has been pre-coated with an antibody against a surface antigen expressed by the target cell line and measuring the fluorescence inside the immune cells or quantifying the number of engulfed beads or cells)). Methods for determining attenuation of macrophage cytolytic function and phagocytosis of allogeneic graft 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 "masking" or "masked" genes.

[0110] As used herein, the term "inducing apoptosis of leukocytes attacking the graft" refers to a transgene encoding a gene product that functions to kill leukocytes attacking the graft in the vicinity of allogeneic graft cells (Huang et al., Proc Natl Acad Sci USA. 96:14871-6 (1999); Suzuki et al., Proc Natl Acad Sci USA. 97:1707-12 (2000); Simon et al., Proc Natl Acad Sci USA. 98:5158-63 (2001)). In one embodiment, inducing apoptosis of leukocytes attacking the graft refers to an increase in apoptosis of leukocytes attacking the graft of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control (e.g., as determined using an assay for apoptosis such as TUNEL staining, caspase staining or annexin-V staining or using fluorescence viability staining). Methods for determining induction of apoptosis of leukocytes attacking the graft are known in the art. Examples of gene products that can induce apoptosis of leukocytes attacking the graft include, but are not limited to: FASLG (FasL) and Tnfsf10. Such transgenes may be referred to herein as "masking" or "masked" genes.

[0111] As used herein, the term "attenuating local inflammatory proteins" refers to a transgene encoding a gene product that functions to inhibit the activity of a local protein, wherein the function of the protein is to promote the accumulation of leukocytes attacking the graft and / or their cytolytic function (Felix et al., Nat Rev Immunol. 17:112-29 (2017)). In one embodiment, attenuation of local inflammatory proteins refers to a decrease in local inflammatory proteins of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control (e.g., as determined using an assay for inflammatory proteins that promote leukocyte activation or migration to the site of inflammation (e.g., chemokines such as CCL2, CCL3, CCL5, CXCL1, CXCL2 and CXCL8 or pro-inflammatory 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 attenuation 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 "masking" or "masked" genes.

[0112] As used herein, the term "protection from leukocyte-mediated apoptosis" refers to a transgene encoding a gene product that functions to inhibit any cellular component that can induce apoptosis or cytolysis of allogeneic graft cells (Abdullah et al., J Immunol. 178:3390-9 (2007)). In one embodiment, protection from leukocyte-mediated apoptosis means that leukocyte-mediated apoptosis is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control (e.g., as determined using an assay for 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 a fluorescent viability stain or by measuring the secretion of cytolytic granules (e.g., perforin, granzyme or other cytolytic proteins released from immune cells) released from the leukocytes). Methods for determining protection from leukocyte-mediated apoptosis are known in the art. Examples of gene products that provide protection from leukocyte-mediated apoptosis include, but are not limited to: Serpin B9 (Spi6) and Dad1. Such transgenes may be referred to herein as "masking" or "masked" genes.

[0113] As used herein, the term "biological agent" refers to a designed polypeptide and the 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 one embodiment, agonist function is a function that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the function relative to a control. In one embodiment, antagonist function is a function that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the function relative to a control.

[0114] As used herein, the term "operably linked" means that a first molecule is linked to a second molecule, where the molecules are 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, if a promoter regulates the transcription of a target transcribable polynucleotide molecule in a cell, the promoter is operably linked to the transcribable polynucleotide molecule. Additionally, if two portions of a transcriptional regulatory element are linked together such that the transcriptional activation functionality of one portion is not adversely affected by the presence of the other portion, the two portions are operably linked to each other. Two transcriptional regulatory elements may be operably linked to each other by a linker nucleic acid (e.g., an intervening non-coding nucleic acid), or may be operably linked to each other in the absence of an intervening nucleotide.

[0115] As used herein, the term "promoter" means a recognition site on DNA to which RNA polymerase binds. The polymerase drives the transcription of a transgene.

[0116] The "percent sequence identity (%)" relative 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 the nucleic acids or amino acids 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 achieved in various ways within the capabilities of one of ordinary skill in the art, such as using publicly available computer software, such as BLAST, BLAST-2, or Megalign software. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. For example, the sequence comparison computer program BLAST can be used to generate percent sequence identity values. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which can alternatively be phrased as a given nucleic acid or amino acid sequence A has or contains a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows:

[0117] 100×(fraction X / Y)

[0118] where X is the number of nucleotides or amino acids scored as identical matches in the alignment of A and B by a sequence alignment program (e.g., BLAST), and where Y is the total number of nucleic acids in B. It should be understood that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0119] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent that is to be administered to a subject, such as a mammal (e.g., a human), to prevent, treat, or control a specific disease or disorder that affects or may affect the subject, the therapeutic agent optionally being combined with one or more pharmaceutically acceptable excipients, diluents, and / or carriers.

[0120] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, and / or other problem complications, and are commensurate with a reasonable benefit / risk ratio.

[0121] As used herein, the term "wild-type" refers to the genotype with the highest frequency for a particular gene in a given organism.

[0122] As used herein, the terms "cell division locus", "cell division loci", and "CDL" refer to genomic locus (or loci) whose transcripts are expressed by dividing cells. When the CDL contains a single locus, the absence of CDL expression in a cell (or its derivatives) means that tumor initiation and / or formation is prohibited because the cell will be eliminated in the absence of CDL expression or because the proliferation of the cell will be blocked or impaired in the absence of CDL expression. When the CDL contains multiple loci, the absence of expression of all or a subset of the loci in a cell (or its derivatives) means that tumor initiation and / or formation is prohibited because the cell will be eliminated in the absence of CDL expression or because the proliferation of the cell will be blocked or impaired in the absence of CDL expression. The CDL may or may not be expressed in non-dividing and / or non-proliferating cells. The CDL may be endogenous to the host cell, or it may be a transgene. If the CDL is a transgene, it may be from the same or a different species as the host cell, or it may have a 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 contains two or more loci that are transcribed during cell division. For example, in one embodiment, the multi-locus CDL contains two MYC genes (c-Myc and N-myc) (Scognamiglio et al., 2016). In one embodiment, the multi-locus CDL contains AURORA B and C kinases that may have overlapping functions (Fernandez-Miranda et al., 2011). Cell division and cell proliferation are terms that may be used interchangeably herein.

[0123] As used herein, the terms "normal rate of cell division", "normal cell division rate", "normal rate of cell proliferation", and "normal cell proliferation rate" refer to the rate of cell division and / or proliferation that is typical of non-cancerous healthy cells. The normal rate of cell division and / or proliferation can be cell type-specific. For example, it is well known that the number of cells in the epidermis, intestine, lung, blood, bone marrow, thymus, testis, uterus, and breast is maintained by high rates of cell division and high rates of cell death. In contrast, the number of cells in the pancreas, kidney, cornea, prostate, bone, heart, and brain is maintained by low rates of cell division and low rates of cell death (Pellettieri and SánchezAlvarado, 2007).

[0124] As used herein, the terms "inducible negative effector of proliferation" and "iNEP" refer to genetic modifications that promote the control of cell division and / or proliferation using CDL expression by: i) inducibly terminating or blocking CDL expression, thereby prohibiting cell division and proliferation; ii) inducibly eliminating at least a portion of the cells expressing CDL (i.e., killing at least a portion of the proliferating cells); or iii) inducibly slowing the rate of cell division relative to the normal cell division rate of the cell such that the rate of cell division will not be fast enough to contribute to tumor formation.

[0125] As used herein, the terms "ablation link" and "ALINK" refer to examples of iNEPs that include a transcriptional link between CDL and a sequence encoding a negative selectable marker. The ALINK modification allows the user to inducibly kill proliferating host cells containing ALINK or inhibit the proliferation of host cells by exposing the ALINK-modified cells to an inducer of the negative selectable marker to kill at least a portion of the proliferating cells. For example, cells modified to contain ALINK at CDL can be treated with an inducer of the negative selectable marker (e.g., a prodrug) in order to ablate proliferating cells or inhibit cell proliferation by killing at least a portion of the proliferating cells.

[0126] As used herein, the terms "CDL-regulated exogenous activator" and "EARC" refer to examples of iNEP that include exogenous altered mechanisms or systems that promote transcription of non-coding or coding DNA or corresponding translation via an activator. EARC modification allows the user to inductively terminate or inhibit the division of cells containing EARC by removing an inducer that permits transcription and / or translation of the CDL that permits EARC modification from the EARC-modified cells. For example, an inducible activator-based gene expression system can be operably linked to a CDL and used to exogenously control the expression or translation of the CDL such that the presence of a drug-inducible activator and the corresponding inducer drug are necessary for CDL transcription and / or translation. In the absence of the inducer drug, cell division and / or proliferation will terminate or be inhibited (e.g., slowed to the normal cell division rate). For example, CDL Cdk1 / CDK1 can be modified to include a dox-bridge such that expression of Cdk1 / CDK1 and thus cell division and proliferation are only possible in the presence of an inducer (e.g., doxycycline).

[0127] As used herein, the term "proliferation antagonist system" refers to a natural or engineered compound whose presence inhibits (fully or in part) the proliferation of cells.

[0128] As used herein, the term "dox-bridge" refers to a mechanism that separates the activity of a promoter from a target transcription region by expressing rtTA (Gossen et al., 1995) via an endogenous or exogenous promoter and enables transcription of the target region under the control of TRE. As used herein, "rtTA" refers to the reverse tetracycline trans-activator element of the tetracycline-inducible system (Gossen et al., 1995), and "TRE" refers to a promoter consisting of TetO operator sequences upstream of a minimal promoter. After binding of rtTA 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 into the same transcription unit as the CDL or into a different location in the genome, as long as the permissive or non-permissive state of transcription expression of the target region is controlled by doxycycline. The dox-bridge is an example of an EARC.

[0129] 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 in one or more CDLs (e.g., at least two, three, four, or five CDLs). A fail-safe cell can contain an ALINK or EARC or both ALINK and EARC modifications (e.g., ALINK and EARC modifications in different CDLs or in a single CDL).

[0130] As used herein, the term "fail-safe" refers to the property of a cell that is unlikely to exhibit uncontrolled (e.g., oncogenic) proliferation. A cell can be considered "fail-safe" when cell proliferation is under the control of negative regulators or inducers and the likelihood of the cell losing the activity of the system that controls proliferation due to genetic mutations is low. The fail-safe capacity will depend on the number of ALINKs and the number of CDLs targeted by the ALINKs (e.g., a homozygously modified cell with two different CDLs has a higher fail-safe capacity (e.g., is less likely to lose all systems that control proliferation through genetic mutations) than a heterozygously modified cell with a single CDL). The fail-safe property is further described in Table 3. BRIEF DESCRIPTION OF THE DRAWINGS

[0131] The patent or application file contains at least one color drawing. Upon request and payment of the necessary fees, this firm will provide a copy of the published patent or patent application with color drawings.

[0132] In the following detailed description with reference to the accompanying drawings, these and other features of the present disclosure will become more apparent, wherein:

[0133] Figures 1A - 1D Depicts representative images showing the expression of the masking 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.

[0134] Figures 2A - 2E Is a flow cytometry plot showing T cell activation using splenocytes( Figure 2A ), wt B16 melanoma cells( Figure 2B ), masked B16 melanoma cells( Figure 2C ), wt ES cells( Figure 2D ), and masked ES cells( Figure 2E ) in the mixed lymphocyte reaction.

[0135] Figures 3A - 3B Is a schematic diagram and image showing that masked( Figure 3A ) B16F10 cancer cells are protected from rejection in an allogeneic model compared to their WT counterparts( Figure 3B ). Representative images of unmasked cells in C57BL / 6 (n = 5) and unmasked cells in FVB / N (n = 4)( Figure 3A ); masked cells in C57BL / 6 (n = 5) and masked cells in FVB / N (n = 6)( Figure 3B ).

[0136] Figure 4 Schematic showing the formation of tumors by shielded embryonic stem cells in syngeneic B6 mouse recipients (upper panel) and FVB allogeneic recipients (lower panel).

[0137] Figures 5A - 5C Series of photographs depicting allogeneic mice bearing teratomas formed by subcutaneous injection of shielded C57BL / 6 ES cells. Red arrows indicate teratomas. Figure 5A Showing teratomas in C3H mice, Figure 5B Showing teratomas in FVB / N mice, Figure 5C Showing teratomas in CD1 mice.

[0138] Figure 6 Schematic and series of images showing that animals with shielded tissue are not immunocompromised.

[0139] Figure 7 Series of images of FVB / N mice showing additional results that animals with shielded tissue are not immunocompromised.

[0140] Figures 8A - 8H Showing transgene expression in embryonic stem cells derived from C57BL / 6 mice containing a cloning safety failure. Figure 8A Showing FasL expression, Figure 8B Showing Ccl21b expression, Figure 8C Showing Cd200 expression, Figure 8D Showing Cd47 expression, Figure 8E Showing Mfge8 expression, Figure 8F Showing Spi6 expression, Figure 8G Showing H2-M3 expression, and Figure 8H Showing PD-L1 expression.

[0141] Figure 9 Series of diagrams depicting shielded transgene expression in ES cell clones. Each shielded transgene is depicted 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 white blood cells isolated from murine lymphoid organs), where the next outer ring represents 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 (represented by red squares) have the highest shielded gene expression. These clones survive in allogeneic hosts.

[0142] Figure 10It is a figure depicting the masking of transgene expression in the whole-genome gene expression level distribution of the entire genome of ES cells. The expression levels of all 8 transgene-masking genes in the NT2 cell line and NT2-derived teratomas are among the top 5% of all genes in the ES cell genome, and the expression levels of 5 transgene-masking genes are among the top 1% of all genes in the ES cell genome. The expression levels of the transgenes in the NT2 line and NT2-derived teratomas successfully achieved allograft tolerance.

[0143] Figures 11A - 11B It is a photograph showing C57BL / 6-derived teratomas in FVB / N mice. The transgenic line NT2 produced 9 teratomas at 10 injection sites. Images were taken 3 months after injection. Figure 11B It is composed of Figure 11A An enlarged image of the teratoma indicated by the arrow in

[0144] Figures 12A - 12B It is a figure showing the teratoma tumor sizes in syngeneic ( Figure 12A ) and allogeneic ( Figure 12B ) mice treated with ganciclovir.

[0145] Figures 13A - 13B It is a series of micrographs showing that the masked embryonic stem cells injected into syngeneic ( Figure 13A ) and allogeneic ( Figure 13B ) hosts can differentiate into all three cell lineages.

[0146] Figures 14A - 14D It is a micrograph showing the formation of all three germ layers in teratomas formed by subcutaneous injection of masked ES cells into mice. Figure 14A , Figure 14B and Figure 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, thus confirming that the tissue is well vascularized.

[0147] Figure 15 It is a schematic diagram showing the construction of a vector expressing the target gene essential for allograft tolerance.

[0148] Figures 16A - 16H It is a fluorescence micrograph showing the expression of the protein encoded by the transgene masked 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 16DShows 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.

[0149] Figures 17A - 17B is a micrograph showing that the shielded ES cells have a typical ES cell morphology ( Figure 17A ) and express the ES cell marker alkaline phosphatase ( Figure 17B ).

[0150] Figures 18A - 18B is a fluorescence micrograph showing the expression of markers of pluripotent ES cells (Oct4 ( Figure 18A ) and SSEA1 ( Figure 18B )) in the shielded ES cells. Figures 18A - 18B The inset in shows single-channel images of fluorescence micrographs of ES cell markers (Oct4 and SSEA) and DAPI labeling the cell nuclei, to demonstrate the co-localization of the staining of ES cell markers with the shielded cells.

[0151] Figure 19 is a schematic diagram depicting the immune process of suppression in ES cells by shielding a transgene (upper panel) and an expression cassette for expressing the shielded transgene (lower panel).

[0152] Figure 20 is a series of graphs depicting the effect of interferon γ (IFNγ) on the MHC levels in ES cells. IFNγ increases the MHC levels in wild-type ES cells and ES cells overexpressing the wild-type IFNγ receptor IFNγR1, but does not increase the MHC levels in ES cells overexpressing the dominant negative form of the IFNγ receptor (IFNγR1 d39), thereby indicating that IFNγR1 d39 completely inhibits the IFNγ-mediated upregulation of MHC in ES cells. Detailed Description

[0153] Description of Cells and Methods

[0154] Characterized by tools such as genetically modified cells, and methods for providing local immune containment at the transplantation site using such cells, for example, when transplanting the cells into an allogeneic host. The genetically modified cells comprise: one or a group of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane-bound or locally acting and whose function is to mitigate the function of the host immune system (e.g., activation of leukocytes and NK cells that attack the graft) or to act as a defense mechanism against attacking leukocytes.

[0155] A variety of cytoplasmic, membrane-bound, or locally acting immune factors have been found to regulate local immune compartments and local immune populations. Immune factors such as PD-L1 (Brown et al., J Immunol. 170:1257-66 (2003); Curiel et al., Nat Med. 9:562-7 (2003); Dong et al., Nat Med. 8:793-800 (2002)), CD47 ((Willingham et al., Proc Natl Acad Sci USA. 109:6662-7 (2012); Liu et al., PLoS One. 10:e0137345 (2015); Demeure et al., J Immunol. 164:2193-9 (2000)), CD200 (Jenmalm et al., J Immunol. 176:191-9 (2006); Cherwinski et al., J Immunol. 174:1348-56 (2005); Kretz-Rommel et al., J Immunol. 178:5595-605 (2007)), FasL (O'Connell et al., J Exp Med. 184:1075-82 (1996); Ju et al., Nature. 373:444-8 (1995); Mazar et al., J Biol Chem. 284:22022-8 (2009)), and Spi6 (Medema Proceedings of the National Academy of Sciences of the United States of America. 98:11515-20 (2001); Zhang et al., Immunity. 24:451-61 (2006); Soriano et al., Lung Cancer. 77:38-45 (2012)) among many others that have been described, including their roles in immune regulation. Expression of one or more of these immune regulatory factors in allogeneic cells has been found to be useful for providing local immune containment and / or reducing allograft rejection in a host to which the cells are administered.

[0156] Allogeneic cells are modified by using specific immunomodulatory factors introduced into the cells or cell populations. The modified cells evade immune rejection by simultaneously modulating many different local immune pathways. Regardless of the genetic background, such genetically engineered cells can be "off-the-shelf" transplanted into many recipients and are not rejected by the recipient's immune system. This immunomodulatory approach overcomes the requirement for systemic immune containment of the transplant recipient, which can be dangerous to the recipient. Thus, although immunosuppressive agents can be administered to patients receiving the modified cells described herein, the therapy does not necessarily include the administration of immunosuppressive agents. This immunomodulatory approach also overcomes expensive and impractical methods of obtaining patient-specific iPS cells, manipulating regulatory cells, or inducing chimerism by hematopoietic cell transplantation (HCT).

[0157] Cells can be genetically modified to express a set of transgenes that encode gene products that are cytoplasmic, membrane-bound, or locally acting and whose function is to attenuate immune function (e.g., activation of white blood cells and NK cells that attack the graft) or act as a defense matrix against immune responses (e.g., attack of white blood cells). The set of transgenes can be selected from genes that have a role in the above-described immunomodulatory pathways. Such genes include, but are not limited to, those provided in Table 1.

[0158] Table 1: Genes expressed by allogeneic cells for local immune containment

[0159]

[0160] C-C motif chemokine ligand 21 (Ccl21) is expressed by local lymph nodes, where it is used to attract activated antigen-presenting cells (APCs). This key function provides the opportunity to "reverse" APC migration by overexpressing this gene on the transplanted cells. Indeed, some melanomas express Ccl21 and recruit CCR7 + cells, which in turn can reorganize part of their tumor stroma into "self". This leads to stromal remodeling that directs the recruitment and maintenance of Cd4 + Tregs (Zindl et al., Science. 328:697-8 (2010)). Indeed, expression of Ccl21 on tumors can protect Ccl21-deficient tumor cells co-implanted in a syngeneic allograft environment from rejection (Shields et al., Science. 328:749-52 (2010)). Ccl21b is the murine ortholog of human Ccl21.

[0161] The amino acid sequences of murine and human Ccl21 are:[[]]

[0162] Murine Ccl21

[0163] MAQMMTLSLLSLVLALCIPWTQGSDGGGQDCCLKYSQKKIPYSIVRGYRKQEPSLGCPIPAILFLPRKHSKPELCANPEEGWVQNLMRRLDQPPAPGKQSPGCRKNRGTSKSGKKGKGSKGCKRTEQTQPSRG(SEQ ID NO:1)

[0164] Human Ccl21

[0165] MAQSLALSLLILVLAFGIPRTQGSDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIP

[0166] AILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGP(SEQ ID NO:2)

[0167] Expression of Cd47 in umbilical cord blood promotes the development of hyporesponsive T cells (Avice et al., J Immunol. 167:2459-68 (2001)). Since they lack "self" presentation, red blood cells also upregulate Cd47 to avoid dendritic cell activation (vanden Berg et al., Immunity. 43:622-4 (2015)). Recently, it was shown that increased expression of human Cd47 enhances engraftment in a murine model of porcine-to-human hematopoietic cell transplantation (Tena et al., Am J Transplant. 14:2713-22 (2014)).

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

[0169] Murine Cd47

[0170] MWPLAAALLLGSCCCGSAQLLFSNVNSIEFTSCNETVVIPCIVRNVEAQSTEEMFVKWKLNKSYIFIYDGNKNSTTTDQNFTSAKISVSDLINGIASLKMDKRDAMVGNYTCEVTELSRE

[0171] GKTVIELKNRTVSWFSPNEKILIVIFPILAILLFWGKFGILTLKYKSSHTNKRIILLLVAGLVLTVIVVVGAILLIPGEKPVKNASGLGLIVISTGILILLQYNVFMTAFGMTSFTIAILITQVLGYVLALVGLCLCIMACEPVHGPLLISGLGIIALAELLGLVYMKFVASNQRTIQPPRNR(SEQ ID NO:3)

[0172] Human Cd47

[0173] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE(SEQ ID NO:4)

[0174] Cd200 is also an important immune regulatory molecule; increased expression can reduce the severity of allograft rejection, autoimmunity, and allergic diseases (Gorczynski et al., J Immunol. 172:7744-9 (2004)). It has been shown that, in vitro, APC expression of Cd200 inhibits the production of interferon γ (IFN-γ) and lytic granules by activated Cd8+ T cells (Misstear et al., J Virol. 86:6246-57 (2012)). Most intriguingly, overexpression of Cd200 increases the survival of skin and heart allografts in mice by promoting Foxp3+ Treg cells (Gorczynski et al., Transplantation. 98:1271-8 (2014)).

[0175] The amino acid sequences of murine and human Cd200 are as follows:

[0176] Murine Cd200

[0177] MGSLVFRRPFCHLSTYSLIWGMAAVALSTAQVEVVTQDERKALHTTASLRCSLKTSQEPLIVTWQKKKAVSPENMVTYSKTHGVVIQPAYKDRINVTELGLWNSSITFWNTTLEDEGCYMCLFNTFGSQKVSGTACLTLYVQPIVHLHYNYFEDHLNITCSATARPAPAISWKGTGTGIENSTESHFHSNGTTSVTSILRVKDPKTQVGKEVICQVLYLGNVIDYKQSLDKGFWFSVPLLLSIVSLVILLVLISILLYWKRHRNQERGESSQGMQRMK(SEQ ID NO:5)

[0178] Human Cd200

[0179] MERLVIRMPFSHLSTYSLVWVMAAVVLCTAQVQVVTQDEREQLYTPASLKCSLQNAQEALIVTWQKKKAVSPENMVTFSENHGVVIQPAYKDKINITQLGLQNSTITFWNITLEDEGCYMCLFNTFGFGKISGTACLTVYVQPIVSLHYKFSEDHLNITCSATARPAPMVFWKVPRSGIENSTVTLSHPNGTTSVTSILHIKDPKNQVGKEVICQVLHLGTVTDFKQTVNKGYWFSVPLLLSIVSLVILLVLISILLYWKRHRNQDRGELSQGVQKMT(SEQ ID NO:6)

[0180] 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) evade immune rejection by upregulating this molecule (El Haddad et al., Blood. 117:1176-83 (2011)). It has also recently been demonstrated that the ability of dendritic cells to present antigen to cytotoxic T cells without being killed by contact-mediated cytotoxicity is mediated by Spi6 (Lovo et al., J Immunol. 188:1057-63 (2012)). Spi6 is also known as Serpin B9.

[0181] The amino acid sequences of murine Spi6 and its human counterpart Serpin B9 are:

[0182] Mouse Spi6

[0183] MNTLSEGNGTFAIHLLKMLCQSNPSKNVCYSPASISSALAMVLLGAKGQTAVQISQALGL

[0184] NKEEGIHQGFQLLLRKLNKPDRKYSLRVANRLFADKTCEVLQTFKESSLHFYDSEMEQLSFAEEAEVSRQHINTWVSKQTEGKIPELLSGGSVDSETRLVLINALYFKGKWHQPFNKEYTMDMPFKINKDEKRPVQMMCREDTYNLAYVKEVQAQVLVMPYEGMELSLVVLLPDEGVDLSKVENNLTFEKLTAWMEADFMKSTDVEVFLPKFKLQEDYDMESLFQRLGVVDVFQEDKADLSGMSPERNLCVSKFVHQSVVEINEEGTEAAAASAIIEFCCASSVPTFCADHPFLFFIRHNKANSILFCGRFSSP(SEQ ID NO:7)

[0185] Human Serpin B9

[0186] METLSNASGTFAIRLLKILCQDNPSHNVFCSPVSISSALAMVLLGAKGNTATQMAQALSL

[0187] NTEEDIHRAFQSLLTEVNKAGTQYLLRTANRLFGEKTCQFLSTFKESCLQFYHAELKELS

[0188] FIRAAEESRKHINTWVSKKTEGKIEELLPGSSIDAETRLVLVNAIYFKGKWNEPFDETYTREMPFKINQEEQRPVQMMYQEATFKLAHVGEVRAQLLELPYARKELSLLVLLPDDGVELS

[0189] TVEKSLTFEKLTAWTKPDCMKSTEVEVLLPKFKLQEDYDMESVLRHLGIVDAFQQGKADLSAMSAERDLCLSKFVHKSFVEVNEEGTEAAAASSCFVVAECCMESGPRFCADHPFLFFIRHNRANSILFCGRFSSP(SEQ IDNO:8)

[0190] Activated, cytotoxic Cd8+ T cells can kill target cells through the expression of FasL, which binds to the FAS receptor and activates caspase-mediated apoptosis of the targeted cells. However, many tumors develop 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 murine solid tumors has been associated with sparse Cd8+ T cell infiltration and abundant FoxP3+ Treg cells (Motz et al., Nat Med. 20:607-15 (2014)). Recently, it has been shown that B lymphocytes also use the expression of FasL to kill T helper cells during the effector phase of the immune response (Lundy et al., Front Immunol. 6:122 (2015)). FasL is the murine ortholog of human FASLG.

[0191] The amino acid sequences of murine FasL and its human counterpart FASLG are:

[0192] Murine FasL

[0193] MQQPMNYPCPQIFWVDSSATSSWTPPGSVFPCPSSGPRGPDQRRPPPPPPPVSPLPPPSQPLPLPPLTPLKKKDHNTNLWLPVVFFMVLVALVGMGLGMYQLFHLQKELAELREFTNQSLKVSSFEKQIANPSTPSEKKELRSVAHLTGNPHSRSIPLEWEDTYGTALISGVKYKKGSLVINEAGLYFVYSKVYFRGQSCNNQPLNHKVYMRNSKYPGDLVLMEEKRLNYCTTGQIWAHSSYLGAVFNLTSADHLYVNISQLSLINFEESKTFFGLYKL(SEQ ID NO:9)

[0194] Human FASLG

[0195] MQQPFNYPYPQIYWVDSSASSPWAPPGTVLPCPTSVPRRPGQRRPPPPPPPPPLPPPPPPPPLPPLPLPPLKKRGNHSTGLCLLVMFFMVLVALVGLGLGMFQLFHLQKELAELRESTSQMHTASSLEKQIGHPSPPPEKKELRKVAHLTGKSNSRSMPLEWEDTYGIVLLSGVKYKKGGLVINETGLYFVYSKVYFRGQSCNNLPLSHKVYMRNSKYPQDLVMMEGKMMSYCTTGQMWARSSYLGAVFNLTSADHLYVNVSELSLVNFEESQTFFGLYKL(SEQ ID NO:10)

[0196] PD-L1 is a key immunomodulatory molecule that binds to programmed cell death 1 (PD-1). PD-1 is expressed on T cells, and binding to PD-L1 results in T cell anergy (MacDonald et al., J Immunol. 126:1671-5 (1981)).

[0197] The amino acid sequences of murine and human PD-L1 are:

[0198] Murine PD-L1

[0199] MRIFAGIIFTACCHLLRAFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWEKE

[0200] DEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGKRSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNRTHWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET(SEQ ID NO:11)

[0201] Human PDL1 (CD274)

[0202] MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET(SEQ ID NO:12)

[0203] Like those induced by allografts, the inflammatory environment attracts macrophages and inflammatory monocytes, as well as many other innate immune cells. Milk fat globule epidermal growth factor–8 (Mfge-8) is expressed in many murine tumors (Neutzner et al., Cancer Res. 67:6777-85 (2007)) and has been shown to contribute to local immune containment by polarizing incoming monocytes into inhibitory M2-like macrophages (Soki et al., J Biol Chem. 289:24560-72 (2014)).

[0204] The amino acid sequences of murine and human MFGE-8 are:

[0205] Murine MFGE8

[0206] MQVSRVLAALCGMLLCASGLFAASGDFCDSSLCLNGGTCLTGQDNDIYCLCPEGFTGLVCNETERGPCSPNPCYNDAKCLVTLDTQRGDIFTEYICQCPVGYSGIHCETETNYYNLDGEYMFTTAVPNTAVPTPAPTPDLSNNLASRCSTQLGMEGGAIADSQISASSVYMGFMGLQRWGPELARLYRTGIVNAWTASNYDSKPWIQVNLLRKMRVSGVMTQGASRAGRAEYLKTFKVAYSLDGRKFEFIQDESGGDKEFLGNLDNNSLKVNMFNPTLEAQYIKLYPVSCHRGCTLRFELLGCELHGCSEPLGLKNNTIPDSQMSASSSYKTWNLRAFGWYPHLGRLDNQGKINAWTAQSNSAKEWLQVDLGTQRQVTGIITQGARDFGHIQYVASYKVAHSDDGVQWTVYEEQGSSKVFQGNLDNNSHKKNIFEKPFMARYVRVLPVSWHNRITLRLELLGC(SEQ ID NO:13)

[0207] Human MFGE8

[0208] MPRPRLLAALCGALLCAPSLLVALDICSKNPCHNGGLCEEISQEVRGDVFPSYTCTCLKGYAGNHCETKCVEPLGMENGNIANSQIAASSVRVTFLGLQHWVPELARLNRAGMVNAWTPSSNDDNPWIQVNLLRRMWVTGVVTQGASRLASHEYLKAFKVAYSLNGHEFDFIHDVNKKHKEFVGNWNKNAVHVNLFETPVEAQYVRLYPTSCHTACTLRFELLGCELNGCANPLGLKNNSIPDKQITASSSYKTWGLHLFSWNPSYARLDKQGNFNAWVAGSYGNDQWLQVDLGSSKEVTGIITQGARNFGSVQFVASYKVAYSNDSANWTEYQDPRTGSSKIFPGNWDNHSHKKNLFETPILARYVRILPVAWHNRIALRLELLGC(SEQ ID NO:14)

[0209] The potent killing potential of NK cells is also crucial in transplant rejection. NK cells can kill target cells lacking MHC class I molecules, as well as other cells in an inflammatory environment. H2-M3, the murine homolog of human HLA-G, has recently been shown to have a regulatory effect on NK cells, allowing them to ignore cells lacking "self molecules" (Andrews et al., Nat Immunol. 13:1171-7 (2012)). This is thought to be achieved by binding HLA-G, an immune inhibitory receptor, on NK cells and T cells (Carosella et al., Adv Immunol. 127:33-144 (2015)). H2-M3 is the murine ortholog of human HLA-G.

[0210] The amino acid sequences of murine H2-M3 and its human counterpart HLA-G are:

[0211] Murine H2-M3

[0212] SIEEIPRMEPRAPWMEKERPEYWKELKLKVKNIAQSARANLRTLLRYYNQSEGGSHILQWMVSCEVGPDMRLLGAHYQAAYDGSDYITLNEDLSSWTAVDMVSQITKSRLESAGTAEYFRAYVEGECLELLHRFLRNGKEILQRADPPKAHVAHHPRPKGDVTLRCWALGFYPADITLTWQKDEEDLTQDMELVETRPSGDGTFQKWAAVVVPSGEEQRYTCYVHHEGLTEPLALKWGRSSQSSVVIMV (SEQ ID NO:15)

[0213] Human HLA-G

[0214] MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD(SEQ ID NO:16)

[0215] A group of transgenes including one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6A can be expressed in cells. The cells can be, for example, stem cells or cells suitable for genome editing, such as cells that can be used for treatment and / or differentiated into therapeutic cell types. The stem cells can be, for example, embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. The group of transgenes can include 1, 2, 3, 4, 5, 6, 7, or all 8 of these genes, or can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 of these genes. The cells can 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. Methods known to those skilled in the art can be used to modify the TGF-β transgene so that the gene product is expressed in a membrane-bound form (i.e., such that the gene product is expressed on the cell surface of an allograft). For example, a method for localizing TGF-β to the membrane is to co-express TGF-β with an additional transgene encoding the LRRC32 protein or any other polypeptide that causes TGF-β to localize to the cell membrane. This protein anchors TGF-β to the membrane. (Tran DQ et al., Proc Natl Acad Sci U.S.A. 106:13445-50 (2009)).

[0216] The amino acid sequence of IFNγR1 d39 is:

[0217] MGPQAAAGRMILLVVLMLSAKVGSGALTSTEDPEPPSVPVPTNVLIKSYNLNPVVCWEYQNMSQTPIFTVQVKVYSGSWTDSCTNISDHCCNIYGQIMYPDVSAWARVKAKVGQKESDYARSKEFLMCLKGKVGPPGLEIRRKKEEQLSVLVFHPEVVVNGESQGTMFGDGSTCYTFDYTVYVEHNRSGEILHTKHTVEKEECNETLCELNISVSTLDSRYCISVDGISSFWQVRTEKSKDVCIPPFHDDRKDSIWILVVAPLTVFTVVILVFAYWYTKKNSFKRKSIMLPKSLLSVVKSATLETKPESKYSLVTPHQPAVLESETVICEEPLSTVTAPDSPEAAEQEELSKETKALEAGGSTSAMTPDSPPTPTQRRSFSLLSSNQSGPCSLTAYHSRNGSDSGLVGSGSSISDLESLPNNNSETKMAEHDPPPVRKA(SEQ ID NO:17)

[0218] These genes can be human genes or murine genes. In one embodiment, the gene is of the same species as the recipient of the allograft in which the cells are to be transplanted. In one embodiment, the gene is of any species in which the function of the gene is conserved or in which the engineered biological agent has agonist function with an endogenous counterpart. Methods for introducing and expressing these transgenes in cells are described herein and are also known to those of skill in the art. Cells expressing these transgenes can be referred to as "cloaked" due to their ability to evade allograft rejection without systemic immune containment and without the need for immunosuppressive drugs.

[0219] It is contemplated herein that a cell population derived from the above-described cloaked cells can also be used to generate local immune containment when transplanted at the transplant site of an allogeneic recipient.

[0220] Before or after generating the shielded cells of the present disclosure, the cells may first be modified to be fail-safe cells. Fail-safe cells use a cell division locus (CDL) to control cell proliferation in animal cells. As provided herein, a CDL is a locus whose transcriptional product is expressed during cell division. As described herein, the CDL may be genetically modified to include a negative selectable marker and / or an inducer-based gene expression system, which allows the user to permit, eliminate, and / or inhibit the proliferation of the genetically modified cells by adding or removing an appropriate inducer. Methods for preparing and using fail-safe cells are described, for example, in WO 2016 / 141480, the entire teachings of which are incorporated herein by reference. The cells may first be made fail-safe and then shielded. Alternatively, the cells may first be shielded and then made fail-safe.

[0221] The cells can be vertebrate cells, such as mammalian cells, such as human cells or mouse cells. The cells can also be vertebrate stem cells, such as mammalian stem cells, such as human stem cells or mouse stem cells. Preferably, the cells or stem cells are suitable for genetic modification. Preferably, the user deems the cells or stem cells to have therapeutic value, meaning that the cells or stem cells can be used to treat a disease, disorder, defect, or injury of a subject in need of treatment for the disease, disorder, defect, or injury.

[0222] In some embodiments, the cells are stem cells or progenitor cells (e.g., iPSCs, embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, pluripotent stem cells, amniotic stem cells, umbilical cord blood stem cells, or neural stem or progenitor cells). In some embodiments, the stem cells are adult stem cells (e.g., somatic stem cells or tissue-specific stem cells). In some embodiments, the stem cells or progenitor cells are capable of differentiating (e.g., the stem cells are totipotent, pluripotent, or multipotent). In some embodiments, the cells are isolated from embryonic or neonatal tissue. In some embodiments, the cells are fibroblasts, monocyte precursors, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, progenitor cells, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroleukemia cell line, osteocytes, synoviocytes, tenocytes, ligament cells, meniscus cells, adipocytes, dendritic cells, or natural killer cells. In some embodiments, the cells are manipulated (e.g., transformed or differentiated) into myocytes, erythro-megakaryocytes, eosinophils, iPS cells, macrophages, T cells, pancreatic beta cells, neurons, cardiomyocytes, blood cells, endocrine progenitor cells, exocrine progenitor cells, duct cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, cholangiocytes, or brown adipocytes. In some embodiments, the cells are myocytes (e.g., skeletal, smooth, or cardiac muscle cells), erythro-megakaryocytes, eosinophils, iPS cells, macrophages, T cells, pancreatic beta cells, neurons, cardiomyocytes, blood cells (e.g., red blood cells, white blood cells, or platelets), endocrine progenitor cells, exocrine progenitor cells, duct cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, cholangiocytes, or white or brown adipocytes.In some embodiments, the cells are hormone-secreting cells (e.g., cells that secrete insulin, oxytocin, endorphin, 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 gland secretory epithelial cells, thyroid epithelial cells, keratinized epithelial cells, gallbladder epithelial cells or surface epithelial cells of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra or vagina), renal cells, skeletal joint synoviocytes, periosteal cells, bone cells (e.g., osteoclasts or osteoblasts), perichondrial cells (e.g., chondroblasts or chondrocytes), cartilage cells (e.g., chondrocytes), fibroblasts, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amniotic or placental membranes or serosal cells (e.g., serosal cells lining body cavities). In some embodiments, the cells are somatic cells. In some embodiments, the cells are derived from skin or other organs such as the heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach. The cells can be from a human or other mammal (e.g., rodent, non-human primate, bovine or porcine cells). The masked cells contemplated herein can be used in cell-based therapies where it may be desirable to evade allogeneic rejection at the localized transplant site.

[0223] In some embodiments, the masked cells described herein survive in a host without stimulating a host immune response for one week or longer (e.g., one week, two weeks, one month, two months, three months, 6 months, one year, two years, three years, four years, five years or longer, e.g., for the lifetime of the cell and / or its progeny). The cells maintain their expression as long as the masking transgene survives in the host (e.g., if the masking transgene is no longer expressed, the masked cells can be removed by the host's immune system). In some embodiments, the masked cells further express a transgene encoding a protein that permits detection of the masked cells in vivo (e.g., a fluorescent protein such as GFP or other detectable marker).

[0224] It is contemplated herein that a combination of masked cells and fail-safe cells can be used in cell-based therapies where it may be desirable to evade allogeneic rejection at the localized transplant site while also being able to eliminate cells that exhibit an undesired growth rate, whether such cells are generated before or after transplantation of the cells into the host. The combined masking and fail-safe techniques allow for localized immune protection while addressing the risk that the recipient will develop a malignancy due to the cells providing local immune containment.

[0225] Methods for generating masked cells

[0226] The compositions and methods described herein can be used to reduce the rejection of allogeneic cells by masking the expression of transgenes. A variety of methods have been established for delivering proteins to mammalian cells and for stably expressing genes encoding proteins in mammalian cells, and such methods can be used to generate the masked cells described herein.

[0227] Polynucleotides encoding masked proteins or therapeutic agents

[0228] One platform that can be used to achieve therapeutically effective expression of a masked protein or therapeutic agent in mammalian cells is via stable expression of a gene encoding the masked protein or therapeutic agent (e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell, or by episomal concatamer formation in the nucleus of a mammalian cell). The gene is a polynucleotide encoding the primary amino acid sequence of the corresponding protein. To introduce an exogenous gene into a mammalian cell, the gene can be incorporated into a vector. The vector can be introduced into the cell by a variety of methods, including transformation, transfection, transduction, direct uptake, particle bombardment, and by encapsulating the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. Such methods are described in more detail, for example, in 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 which are incorporated herein by reference in their entireties.

[0229] A masked protein or therapeutic agent can also be introduced into a mammalian cell by targeting a vector containing a portion of the gene encoding the masked protein or therapeutic agent to the cell membrane phospholipids. For example, by linking the vector molecule to the VSV-G protein (a viral protein that has an affinity for all cell membrane phospholipids), the vector can be targeted to the phospholipids on the extracellular surface of the cell membrane. Methods well known to those skilled in the art can be used to generate such constructs.

[0230] The recognition and binding of mammalian RNA polymerase to a polynucleotide encoding a masking protein or a therapeutic agent is important for gene expression. Thus, sequence elements that exhibit high affinity for transcription factors that recruit RNA polymerase and promote the assembly of the transcription complex at the transcription start site can be included within the polynucleotide. Such sequence elements include, for example, mammalian promoters, the sequences of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase.

[0231] Polynucleotides suitable for the compositions and methods described herein also include those encoding a masking protein or a therapeutic agent downstream of a mammalian promoter. Promoters that can be used to express a masking protein or a therapeutic agent in mammalian cells include constitutive promoters. Constitutive promoters include the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1α promoter, and the PGK promoter. Alternatively, promoters derived from viral genomes can also be used to stably express these agents in mammalian cells. Examples of functional viral promoters that can be used to promote mammalian expression of these agents include the adenovirus late promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the tk promoter of HSV, the mouse mammary tumor virus (MMTV) promoter, the LTR promoter of HIV, the promoter of Moloney virus, the Epstein-Barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter.

[0232] Once the polynucleotides encoding a masking protein or a therapeutic agent described herein are incorporated into the nuclear DNA of mammalian cells, transcription of such polynucleotides can be induced by methods known in the art. For example, expression can be induced by exposing mammalian cells to external chemical agents, such as agents that modulate the binding of transcription factors and / or RNA polymerase to mammalian promoters and thereby regulate gene expression. Chemical agents can be used, for example, to promote the binding of RNA polymerase and / or transcription factors to mammalian promoters by removing repressor proteins that are bound to the promoter. Alternatively, chemical agents can be used to enhance the affinity of mammalian promoters for RNA polymerase and / or transcription factors such that the transcription rate of genes located downstream of the promoter is increased in the presence of the chemical agent. Examples of chemical agents that enhance polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These agents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells according to established protocols to promote gene expression.

[0233] Other DNA sequence elements that can be included in the 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 the polynucleotide containing the target gene such that the DNA adopts a three-dimensional orientation that is 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 include those that encode a masking protein or therapeutic agent and additionally include mammalian enhancer sequences. Many enhancer sequences are now known from mammalian genes, and examples include enhancers from genes encoding mammalian globin, elastase, albumin, alpha-fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include enhancers derived from viral genetic material capable of infecting eukaryotic cells. Examples include the SV40 enhancer on the back side of the origin of replication (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the back side of the origin of replication, and the adenovirus enhancer. Additional enhancer sequences that activate transcription of eukaryotic genes are disclosed in Yaniv et al., Nature 297:17 (1982). The enhancer can be spliced into the vector containing the polynucleotide encoding the masking protein or therapeutic agent, for example, at the 5' or 3' position of this gene. In a preferred orientation, the enhancer is located on the 5' side of the promoter, which in turn is 5' relative to the polynucleotide encoding the masking protein or therapeutic agent.

[0234] The nucleic acid vectors described herein can include the woodchuck post-transcriptional regulatory element (WPRE). The WPRE acts at the transcriptional level by promoting nuclear export of the transcript and / or by increasing the efficiency of polyadenylation of the nascent transcript, thereby increasing the total amount of mRNA in the cell. Addition of the WPRE to the vector can result in a significant improvement in the level of transgene expression from several different promoters in vitro and in vivo.

[0235] In some embodiments, the nucleic acid vectors for use in the compositions and methods described herein include a reporter gene sequence, which can be used to verify gene expression in specific cells and tissues, for example. Reporter gene 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 other enzymes well known in the art. When associated with regulatory elements that drive their expression, reporter gene sequences provide signals that can be detected by conventional means, including enzymatic, radiographical, colorimetric, fluorescent or other spectroscopic, fluorescence-activated cell sorting assays, and immunoassays, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry. For example, in the case where the marker sequence is the LacZ gene, the presence of the signal-carrying vector is detected by an assay for β-galactosidase activity. When the transgene is green fluorescent protein or luciferase, the signal-carrying vector can be visually measured by color or luminescence in a luminometer.

[0236] Techniques for introducing a transgene into a cell

[0237] Transfection

[0238] Techniques that can be used to introduce a transgene, such as the masking transgene or therapeutic transgene described herein, into a target cell (e.g., a mammalian cell) are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the target cells. Mammalian cells (such as human cells) that are subjected to an external electric field in this way are then prone to take up exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection TM utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection TM and the protocols that can be used to perform this technique are described in detail, for example, in Distler et al., Experimental Dermatology 14:315 (2005) and US2010 / 0317114, the respective disclosures of which are incorporated herein by reference.

[0239] Additional techniques that can be used to transfect target cells include the squeeze-and-perforate method. This technique induces rapid mechanical deformation of the cells to stimulate the uptake of exogenous DNA through membrane pores formed in response to the applied pressure. The advantage of this technique is that no carrier is required to deliver nucleic acids into cells, such as human target cells. Squeeze-and-perforate 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.

[0240] Lipofection represents another technique that can be used to transfect target cells. This method involves loading nucleic acids into liposomes, which typically present cationic functional groups, such as quaternary amines or protonated amines, on the outside of the liposome. Due to the anionic nature of the cell membrane, this promotes electrostatic interactions between the liposome and the cell, which ultimately results in the uptake of exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Patent No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that utilize ionic interactions with the cell membrane to drive the uptake of foreign nucleic acids include contacting the cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with polynucleotides to confer a positive charge that facilitates interaction with the cell membrane include activated dendrimers (described, for example, in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference), polyethyleneimine, and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described in detail, for example, in Gulick et al., Current Protocols in Molecular Biology 40:I: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 because this method utilizes an applied magnetic field to direct the uptake of nucleic acids. This technique is described in detail, for example, in US2010 / 0227406, the disclosure of which is incorporated herein by reference.

[0241] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laser transfection (also known as optical transfection), which is a technique that involves exposing the cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow the polynucleotides to penetrate the cell membrane. The biological activity of this technique is similar to that of electroporation and, in some cases, superior to it.

[0242] Impalefection is another technique that can be used to deliver genetic material to target cells. It relies on the use of nanomaterials such as carbon nanofibers, carbon nanotubes, and nanowires. Needle-like nanostructures are synthesized perpendicular to the surface of the substrate. DNA containing the gene intended for intracellular delivery is attached to the surface of the nanostructure. A chip with an array of these needles is then pressed against the cell or tissue. The cells pierced 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.

[0243] 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 of completely biodegradable iron oxide and are coated with specific cationic proprietary molecules that vary according to the application. Their association with gene carriers (DNA, siRNA, viral vectors, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells under 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.

[0244] Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, which is a technique that involves using sound (usually at ultrasonic frequencies) to alter the permeability of the cytoplasmic membrane, permeabilize the cells, and allow 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.

[0245] Microbubbles represent another potential mediator that can be used to modify the genome of target cells according to the methods described herein. For example, microbubbles induced by co-overexpression of the glycoprotein VSV-G with, for example, a genome-modifying protein such as a nuclease can be used to efficiently deliver the protein into cells, which subsequently catalyze site-specific cleavage of an endogenous polynucleotide sequence to prepare the cell's genome for covalent incorporation of a target polynucleotide, 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]. In: Methylation changes in early embryonic genes in cancer [Abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; May 13, 2015, abstract number 122.

[0246] Viral infection

[0247] In addition to achieving high transcription and translation rates, stable expression of foreign genes in mammalian cells can also be achieved by integrating the polynucleotide containing the gene into the nuclear genome of mammalian cells. A variety of vectors have been developed for delivering and integrating polynucleotides encoding foreign proteins into the nuclear DNA of mammalian cells. Examples of expression vectors are disclosed in, for example, WO 1994 / 011026 and are incorporated herein by reference. Expression vectors for use in the compositions and methods described herein contain a masking transgene or a therapeutic transgene, as well as additional sequence elements, for example, for expressing these agents and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express a masking transgene or a therapeutic transgene include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct the transcription of the gene. Other useful vectors for expressing a masking transgene or a therapeutic transgene contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA produced by gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions and polyadenylation signal sites in order to direct efficient transcription of the genes carried on the expression vector. Expression vectors suitable for use in the compositions and methods described herein may also contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics (such as ampicillin, chloramphenicol, kanamycin, or nourseothricin).

[0248] Viral vectors for nucleic acid delivery

[0249] Viral genomes provide a rich source of vectors that can be used to efficiently deliver a target gene into the genome of a target cell (e.g., a mammalian cell, such as a human cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are generally incorporated into the nuclear genome of mammalian cells by either general or specific transduction. These processes occur as part of the natural viral replication cycle and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (e.g., retroviridae viral vectors), adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai virus), 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)). For example, other viruses include noroviruses, togaviruses, flaviviruses, reoviruses, papillomaviruses, hepadnaviruses, human papillomaviruses, human foamy viruses, and hepatitis viruses. Examples of retroviruses include: avian leukosis-sarcoma virus, avian C-type virus, mammalian C-type virus, B-type virus, D-type virus, oncovirus, HTLV-BLV group, lentivirus, alpha-retrovirus, gamma-retrovirus, spumavirus (Coffin, J.M., Retroviridae: The viruses and their replication, Virology, 3rd Edition (Lippincott-Raven, Philadelphia, 1996)). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus, and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030, the disclosure of which is incorporated herein by reference as it relates to viral vectors for gene therapy.

[0250] AAV vectors for nucleic acid delivery

[0251] In some embodiments, the masking transgenes or therapeutic transgenes described herein are incorporated into rAAV vectors and / or viral particles to facilitate their introduction into cells. The rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that comprise (1) a promoter, (2) a heterologous sequence to be expressed (such as the masking transgenes or therapeutic transgenes described herein), and (3) viral sequences that facilitate integration and expression of the heterologous gene. The viral sequences can include those of AAV that are cis - required for DNA replication and packaging (such as functional ITRs) into viral particles. Such rAAV vectors can also contain a marker or reporter gene. Useful rAAV vectors have one or more of the AAVWT genes deleted in whole or in part, but retain functional flanking ITR sequences. The AAV ITR can be of any serotype suitable for a particular application. Methods of using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000), the respective disclosures of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0252] The transgenes and vectors described herein (such as a promoter operably linked to a masking transgene or therapeutic transgene) can be incorporated into rAAV virions to facilitate introduction of the polynucleotide or vector into cells. The capsid proteins of AAV form the outer non - nucleic acid portion of the viral particle and are encoded by the AAVcap gene. The cap gene encodes three viral capsid proteins, VP1, VP2, and VP3, which are required for viral particle assembly. The construction of rAAV viral particles has been described, for example, in US 5,173,414; US 5,139,941; US 5,863,541; US 5,869,305; US 6,057,152; and US 6,376,237; as well as Rabinowitz et al., J. Virol. 76:791 (2002) and Bowles et al., J. Virol. 77:423 (2003), the respective disclosures of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0253] rAAV viral particles that can be used in combination with the compositions and methods described herein include those derived from a variety of AAV serotypes, including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43, and rh74. The construction and use of AAV vectors and AAV proteins of different serotypes are described, for example, in Chao et al., Mol. Ther. 2:619 (2000); Davidson et al., Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al., J. Virol. 72:2224 (1998); Halbert et al., J. Virol. 74:1524 (2000); Halbert et al., J. Virol. 75:6615 (2001); and Auricchio et al., Hum. Molec. Genet. 10:3075 (2001), the disclosures of which are incorporated herein by reference as they relate to AAV vectors for gene delivery.

[0254] Pseudotyped rAAV vectors are also used in combination with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene from a serotype different from the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). Techniques related to the construction and use of pseudotyped rAAV viral particles 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).

[0255] AAV viral particles having mutations within the viral capsid can be more effective than non-mutated capsid viral particles for infecting specific cell types. For example, suitable AAV mutants can have ligand insertion mutations to aid in targeting 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 viral particles that can be used in the methods described herein include those capsid hybrids generated by molecular breeding of viruses and by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

[0256] Genome editing

[0257] In addition to the above, a variety of tools have been developed for incorporating a target gene into a target cell, such as a mammalian cell. One such method for incorporating a polynucleotide encoding a target gene into a target cell involves the use of a transposon. A transposon is a polynucleotide encoding a transposase and containing a target polynucleotide sequence or gene flanked by 5' and 3' excision sites. Once the transposon has been delivered into a cell, expression of the transposase gene begins and produces an active enzyme that cleaves the target gene from the transposon. This activity is mediated by the site-specific recognition of the transposase for the transposon excision sites. In some cases, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from the transposon, the target gene can be integrated into the genome of a mammalian cell by the transposase-catalyzed cleavage of a similar excision site present within the nuclear genome of the cell. This allows the target gene to be inserted at the complementary excision site into the cleaved nuclear DNA, and subsequent covalent ligation to complete the incorporation process by joining the target gene to the DNA of the mammalian cell genome via a phosphodiester bond. In certain cases, the transposon can be a retrotransposon such that the gene encoding the target gene is first transcribed into an RNA product and then reverse transcribed into DNA, followed by incorporation into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (e.g., described in detail in WO 2010 / 085699) and the sleeping beauty transposon (e.g., described in detail in US2005 / 0112764), the respective disclosures of which are incorporated herein by reference as they relate to transposons for gene delivery to target cells.

[0258] Another tool for integrating a target gene into the genome of a target cell is the clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system, which is a system that originally evolved as an adaptive defense mechanism against viral infection in bacteria and archaea. The CRISPR / Cas system includes palindromic repeats within plasmid DNA and the associated Cas9 nuclease. This collection of DNA and protein directs site-specific DNA cleavage of a target sequence by first incorporating foreign DNA into the CRISPR locus. The polynucleotide containing these foreign sequences and the repeat-spacer elements of the CRISPR locus is then transcribed in the host cell to produce guide RNA, which can subsequently anneal to the target sequence and localize the Cas9 nuclease to this site. In this way, highly site-specific Cas9-mediated DNA cleavage can be generated in the foreign polynucleotide because the interaction of Cas9 near the target DNA molecule is controlled by RNA:DNA hybridization. As a result, the CRISPR / Cas system can be designed to cleave any target DNA molecule. This technology has been utilized to edit the eukaryotic genome (Hwang et al., Nature Biotechnology 31:227 (2013)) and can be used as an effective means of site-specifically editing the genome of a target cell in order to cleave DNA prior to incorporating a gene encoding the target gene. The use of CRISPR / Cas to regulate gene expression has been described, for example, in U.S. Patent No. 8,697,359, the disclosure of which is incorporated herein by reference because it relates to the use of the CRISPR / Cas system for genome editing. Alternative methods for site-specific cleavage of genomic DNA prior to incorporation of a target gene into a target cell include the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Unlike the CRISPR / Cas system, these enzymes do not contain a guide polynucleotide for targeting to a specific target sequence. Instead, target specificity is controlled by DNA-binding domains within these enzymes. The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov et al., Nature Reviews Genetics 11:636 (2010); and Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), the respective disclosures of which are incorporated herein by reference because they relate to compositions and methods for genome editing.

[0259] Additional genome editing techniques that can be used to incorporate a polynucleotide encoding a target gene into the genome of a target cell include the use of ARCUS, which can be rationally designed to site-specifically cleave genomic DNA TMMeganucleases. Given the established structure-activity relationships for such enzymes, it is advantageous to use these enzymes to incorporate a gene encoding a target gene into the genome of mammalian cells. Single-chain meganucleases can be modified at certain amino acid positions to produce nucleases that selectively cleave DNA at desired positions, enabling site-specific incorporation of the target gene into the nuclear DNA of target cells. These single-chain nucleases have been extensively described, for example, in U.S. Patent Nos. 8,021,867 and US 8,445,251, the respective disclosures of which are incorporated herein by reference as they relate to compositions and methods for genome editing.

[0260] Masking the expression of a transgene

[0261] The masked 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 masking effect (e.g., an amount sufficient to prevent rejection when injected into a subject, such as a mammalian subject, e.g., a mouse, rat, or human). If subcutaneous injection of the masked cells produces teratomas that are not cleared by the subject's immune system, then transgene expression is considered to produce a masking effect. The masked transgenes are also expressed at a level sufficient to promote the production of the protein encoded by the transgene. Conventional methods known to those of skill in the art (e.g., immunohistochemistry, Western blot analysis, or other methods that allow visualization of the protein) can be used to detect protein production. Preferably, the expression of the masked transgenes is such that all 8 proteins encoded by the masked transgenes (PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6) can be detected in the masked cells (e.g., detected by immunohistochemistry using antibodies against the proteins encoded by the masked transgenes).

[0262] In some embodiments, the masked transgene is expressed in the masked cell at a level similar to the level of endogenous gene expression in activated white blood cells such as T cells (e.g., activated white blood cells from the same species, such as activated white blood cells isolated from lymphoid organs, e.g., expression in masked murine cells is similar to expression in activated white blood cells isolated from murine lymphoid organs). The expression of one or more masked transgenes (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)) is greater than or equal to the expression of the endogenous gene in activated white blood cells (e.g., T cells) from the same species (e.g., the expression level of the masked transgene is equal to the expression level of the endogenous gene in activated white blood cells, or 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more higher than the expression level of the endogenous gene in activated white blood cells). In some embodiments, all 8 masked transgenes are expressed at a level greater than or equal to the expression level of the endogenous gene in activated white blood cells from the same species. Activated white blood cells can be isolated from lymphoid organs, or white blood cells such as T cells can be activated in vitro using anti-CD3 / CD28 beads or other methods employed by those skilled in the art (see, e.g., Frauwith and Thompson, J. Clin Invest 109:295-299 (2002); and Trickett and Kwan, J Immunol Methods 275:251-255 (2003)). The transgene expression in the masked cell can also be compared to the gene expression levels reported in profiling studies of activated T cells (see, e.g., Palacios et al., PLOS one 2:e1222 (2007)). In some embodiments, the masked transgene expression is compared to the expression of the endogenous gene in the wild-type form of the cell (e.g., a stem cell, such as an embryonic stem cell from the same species as the masked cell). The expression of one or more masked transgenes (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)) is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1,000-fold or more higher in the masked cell compared to the expression of the endogenous gene in an unmodified wild-type cell of the same cell type as the masked cell (e.g., a stem cell, such as an embryonic stem cell from the same species).In some embodiments, all eight masking transgenes are expressed at a level greater than the expression level of the endogenous gene in the wild-type form of the cell (e.g., a stem cell, such as an embryonic stem cell from the same species as the masked cell) (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100-fold or more higher). Gene expression can be evaluated by direct comparison with isolated ES cells or by comparison with stem cell expression (e.g., ES cell expression) in the Project Grandiose dataset (www.stemformatics.org / project_grandiose). Techniques known in the art (e.g., quantitative polymerase chain reaction (qPCR)) can be used to measure gene expression.

[0263] Method for providing local immune containment at the transplant site

[0264] It is also characterized by a method for providing local immune containment at the transplant site.

[0265] The method includes providing cells; and expressing a set of transgenes in the cells, each transgene encoding a gene product that is cytoplasmic, membrane-bound, or locally acting and whose function is to reduce the activation of white blood cells and NK cells that attack the graft or to act as a defense mechanism against attacking white blood cells.

[0266] 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 one embodiment, the set of transgenes includes Pd-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6.

[0267] 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 one embodiment, TGF-β or the biological agent is locally acting.

[0268] Techniques for introducing various genetic modifications such as transgenes into animal cells are described herein and are generally known in the art.

[0269] In one embodiment of the method, the cells are a source of stem cells, cells suitable for genome editing, and / or therapeutic cell types (e.g., cells that can differentiate into lineage-restricted cells for cell therapy or cells of a desired target tissue). In one embodiment, 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 progenitors, germline stem cells, lung stem cells or progenitors, 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 progenitors. 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, such as mammalian cells, e.g., human or mouse cells. In some embodiments, cells that are modified to express one or more (e.g., two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are cells in a tissue or organ to be transplanted. In some embodiments, the masked cells (e.g., masked stem cells) are differentiated in vitro into a tissue or organ for transplantation using methods known to those skilled in the art.

[0270] In some embodiments, one million to one hundred billion masked cells (e.g., 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8, 6 x 10 8 , 7 x 10 8 , 8 x 10 8 , 9 x 10 8 , 1 x 10 9 , 2 x 10 9 , 3 x 10 9 , 4 x 10 9 , 5 x 10 9 , 6 x 10 9 , 7 x 10 9 , 8 x 10 9 , 9 x 10 9 , 1 x 10 10 , 2 x 10 10 , 3 x 10 10 , 4 x 10 10 , 5 x 10 10 , 6 x 10 10 , 7 x 10 10 , 8 x 10 10 , 9 x 10 10 or 1 x 10 11 (masked cells) are administered to the transplant site or near it in a subject, or into an organ or tissue to be transplanted.

[0271] Techniques for transplanting genetically modified cells into the transplant site of an allogeneic host are described herein and are generally known in the art.

[0272] Expression of a therapeutic agent by masked cells

[0273] The masked 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 the wild-type form of a protein that is lacking in the subject, such as a protein that has been mutated in the subject's cells or produced in insufficient amounts (e.g., produced at low levels or not produced). In some embodiments, the therapeutic protein is an inhibitory antibody (e.g., an antibody that blocks or neutralizes protein function). Masked cells can be modified to produce an inhibitory antibody to treat a subject having a disease or disorder associated with overproduction or abnormal production of a protein (e.g., production of the protein by cells that normally do not produce the protein, production of the protein at a time or in a location where it is not normally produced, or production of an excessive amount of the protein) or at risk of developing the disease or disorder. In some embodiments, the therapeutic antibody is an agonist antibody (e.g., an activating antibody). An agonist antibody can act by binding to and activating an endogenous receptor (e.g., inducing or increasing signaling downstream of receptor activation or altering the conformation of the endogenous receptor to an open or active state). Masked cells can be modified to produce an agonist antibody to treat a subject having a disease or disorder associated with activation of a receptor or signaling pathway or at risk of developing the disease or disorder. Masked 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. Masked cells that produce a secreted protein or antibody can be delivered as circulating cells, injected into a tissue, organ, or body part in need of the therapeutic protein or antibody, or injected subcutaneously to produce a masked subcutaneous tissue. Masked cells that produce a transmembrane or membrane-bound protein can be injected at or near the site of endogenous cells responsive to the therapeutic protein.

[0274] In some embodiments, the masked cells described herein provide a wild-type copy of a gene that is mutated in the subject (e.g., the masked 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 a subject having a disease or disorder caused by a mutation in an endogenous gene (e.g., a subject having a metabolic disorder associated with one or more of the mutations described below).

[0275] A list of exemplary therapeutic agents that can be co-administered with or produced by masked cells and the related diseases or disorders that can be treated using these therapeutic agents is provided in Table 2 below.

[0276] Table 2: Exemplary Therapeutic Agents That Can be Co-administered with or Expressed by Masked Cells to Treat Diseases

[0277]

[0278]

[0279]

[0280]

[0281]

[0282] Inducible systems for expressing therapeutic agents

[0283] If continuous administration of a therapeutic agent expressed by shielded cells is required to treat a disease or disorder, a constitutive promoter (e.g., CAG, CMV, or other constitutive promoter) described herein or known to those of skill in the art can be used to express the therapeutic agent. If the therapeutic agent is needed intermittently (e.g., during relapses or flare-ups that occur during the disease or disorder but not when the subject is asymptomatic), the therapeutic agent can be expressed by an inducible promoter that provides the ability to express the therapeutic agent only when needed. One exemplary class of therapeutic agents that can be delivered using an inducible promoter is TNFα inhibitors. TNFα inhibitors are currently used to treat rheumatoid arthritis but are administered only intermittently during flare-ups of joint inflammation because constitutive administration of TNFα can lead to systemic immunosuppression. If shielded cells are modified to express a TNFα inhibitor under the control of an inducible promoter, the shielded cells can be used to deliver TNFα intermittently, thus obviating the need for repeated injections. Other therapeutic agents that have potential adverse effects if administered continuously can also be expressed intermittently using an inducible promoter as described herein. Exemplary inducible expression systems are described below.

[0284] Tetracycline response element

[0285] 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 the target gene (e.g., the therapeutic transgene to be expressed by the shielded cells), typically together with a minimal promoter that has extremely low basal expression. A protein called rtTA (which also needs to be expressed by the shielded cells) binds to the TRE and activates transcription in the presence of tetracycline or doxycycline. When tetracycline or doxycycline is removed, rtTA no longer binds to the TRE and the target gene is no longer expressed. An advanced version of this system, the Tet-On Advanced transactivator (rtTA2 s-M2) and Tet-On 3G may be particularly useful for human therapies because they are human codon-optimized and responsive to low concentrations of doxycycline.

[0286] Light-inducible systems

[0287] Another method for the 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 sheltered cells involve a class of proteins that undergo a conformational change and dimerize in response to blue light. These proteins have been fused to DNA-binding and transcriptional components that have been shown to bind to specific promoter sequences and activate transcription when brought together by exposure to blue light (Wang et al., Nat Methods, 9:266-269, 2012). This method of inducibly activating gene expression can be used to control the production of therapeutic agents in sheltered cells administered subcutaneously because blue light can be shone on the skin near the sheltered subcutaneous tissue to induce the sheltered cells to produce the therapeutic agent.

[0288] Radiogenetics

[0289] A third method for the inducible activation of gene expression (e.g., expression of a therapeutic agent by sheltered cells) involves the use of radio waves. In one form of a radio wave-inducible expression system, the TRPV1 receptor is fused to a GFP-binding domain and co-expressed with a form of ferritin linked to GFP (Stanley et al., Nat Med 21:92-98, 2015). The GFP-ferritin binds to the GFP-binding domain of the TRPV1 receptor. When radio waves of a specific frequency are applied to the cells, ferritin interacts with TRPV1 and allows the influx of calcium, which activates the transcription factor NFAT. If a therapeutic agent is operably linked to an NFAT-sensitive promoter element (such as SRE-CRE-NFATRE) and co-expressed with TRPV1-GFP and GFP-ferritin, this system can be used to inducibly express the therapeutic agent. Radio wave-induced expression offers the advantage of being able to induce expression in cells deep within the body because radio waves can penetrate tissue. For example, radiogenetics can be used to regulate gene expression in the retina. Thus, this method can be used to inducibly express a therapeutic transgene in sheltered cells by non-invasive and harmless radio waves.

[0290] Destabilizing domain systems

[0291] Gene expression can also be regulated using destabilizing domain systems. A transgene encoding a target protein (e.g., a therapeutic agent as described herein) can also include a destabilizing domain such that the resulting protein product comprises the target protein fused to the destabilizing domain. Exemplary destabilizing domains include mutants of human FK506- and rapamycin-binding protein (FKBP12) that confer instability to the protein fused to them. FKBP12 mutants include the N-terminal mutants F15S, V24A, H25R, E60G, and L106P, and the 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 destabilizing domains. The destabilizing domain promotes protein degradation. When expression of the target protein (e.g., a therapeutic agent) is desired, a small molecule synthetic ligand can be used to stabilize the protein containing the destabilizing domain. The small molecule ligand Shield-1 (Shld1) can be used to stabilize proteins containing FKBP12 mutants by protecting them from degradation. Other destabilizing domains that can be used to regulate target expressed proteins include mutants of Escherichia coli dihydrofolate reductase (ecDHFR) and mutants of the human estrogen receptor ligand-binding domain (ERLBD) that confer instability and thus degradation when fused to the target protein and can be stabilized by the small molecule ligands 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 respective disclosures of which are incorporated herein by reference as they relate to destabilizing domain systems.

[0292] Coumarate switch-inducible system

[0293] Another method for inducible activation of gene expression involves the use of a coumarate gene switch system. In the repressor configuration of this system, regulation is mediated by the binding of a repressor (CymR) to an operator site (CuO) placed downstream of a strong constitutive promoter. Addition of coumarate, a small molecule, relieves repression, allowing expression of the transgene. Alternatively, a reverse-coumarate-transactivator (rcTA) can be inserted upstream of a minimal CMV promoter, which is operably linked to a transgene encoding a therapeutic agent. A six-fold repeat of the coumarate operator (6xCuO) can be inserted just before the translation start (ATG) of the therapeutic transgene. In the absence of coumarate, rcTA cannot bind to 6xCuO, and thus the transgene encoding the therapeutic agent will not be transcribed because 6xCuO is inactive. When coumarate is added, it forms a complex with rcTA, allowing binding to 6xCuO and transcription of the transgene encoding the therapeutic agent (Mullick et al., 2006).

[0294] Ecdysone-inducible system

[0295] Another example of an inducible gene expression system is the ecdysone-inducible system, in which the N-terminal truncation of the retinoid X receptor (RXR) and the ecdysone receptor (EcR) fused to the activation domain of Vp16 (VpEcR) are inserted into the 5' untranslated region of a gene expressed by a masked cell such that they are co-expressed from an endogenous promoter. An ecdysone response element (EcRE) with a downstream minimal promoter can be inserted immediately upstream of the start codon of a transgene encoding a therapeutic agent. The co-expressed RXR and VpEcR can heterodimerize with each other. In the absence of ecdysone or the synthetic drug analogue muristerone A, the dimerized RXR / VpEcR cannot bind to the EcRE, and thus the transgene encoding the therapeutic agent will not be transcribed. In the presence of ecdysone or muristerone A, the dimerized RXR / VpEcR can bind to the EcRE, such that the transgene encoding the therapeutic agent is transcribed (No et al., 1996). Since ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene.

[0296] Ligand-reversible dimerization system

[0297] In another instance, the transgene encoding the therapeutic agent can be modified such that it is functionally divided into parts / domains, such as a 5' part and a 3' part, and the FKBP peptide sequence can be inserted into each domain. An IRES (Internal Ribosome Entry Site) sequence can be placed between the two domains, which allows for the simultaneous transcription of the two different domains to produce two separate proteins. In the absence of a dimerizing agent, the two separate domains of the therapeutic agent will be functionally inactivated. After the introduction of a dimerizing agent such as rapamycin or AP20187, the FKBP peptides will dimerize, bringing together the 5' and 3' domains of the therapeutic agent and reconstituting the active protein (Rollins et al., 2000).

[0298] Cell-based delivery of therapeutic agents

[0299] Treatment of age-related macular degeneration or retinal dystrophy

[0300] In one example, cells can be modified to produce a VEGF inhibitor, such as a VEGF trap (e.g., a soluble decoy receptor as described in Holash et al., Proc Natl Acad Sci U.S.A. 99:11383-11398, 2002, which is incorporated herein by reference, such as aflibercept) to treat age-related macular degeneration (AMD) or retinal dystrophy. A VEGF trap is a biological agent that binds to and inhibits VEGF, an angiogenic protein that can promote abnormal blood vessel formation. VEGF traps are used to treat wet AMD, which is characterized by abnormal growth of subretinal blood vessels that can lead to retinal detachment and progressive vision loss. To treat AMD, VEGF traps are typically delivered by regular injection into the eye. 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. The masked cells (e.g., stem cells) expressing the VEGF inhibitor (e.g., VEGF trap) can be differentiated into retinal pigment epithelial (RPE) cells using methods known to those skilled in the art prior to administration to the eye, or isolated RPE cells can be modified to express the masked transgene and the VEGF inhibitor. 25,000 to 100,000 masked RPE cells (e.g., 25,000, 50,000, 75,000, or 100,000 masked RPE cells) expressing the VEGF inhibitor (e.g., VEGF trap) can be injected into the subretinal space of each eye to treat wet AMD or retinal dystrophy. 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 inhibitory antibodies (e.g., bevacizumab and ranibizumab).

[0301] Treatment of Parkinson's disease

[0302] In another example, masked cells, such as dopaminergic neurons or cells (e.g., stem cells) that can be differentiated in vitro using methods known to those skilled in the art to produce dopaminergic neurons, can be administered to a subject with Parkinson's disease, which is characterized by the loss of dopaminergic neurons. 25,000 to 100,000 masked dopaminergic neurons (e.g., 25,000, 50,000, 75,000, or 100,000 masked dopaminergic neurons) can be administered to the brain of a subject with Parkinson's disease (e.g., stereotactic injection into the substantia nigra).

[0303] Treatment of myocardial infarction

[0304] The masked 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 part of the heart is reduced or stopped, thereby damaging the heart muscle. To treat a subject with myocardial infarction, masked cells (e.g., stem cells) can be differentiated into cardiomyocytes using methods known to those skilled in the art, or isolated cardiomyocytes can be modified to express a masked transgene. Five hundred million to five billion masked cardiomyocytes (e.g., 5x10 8 、6x10 8 、7x10 8 、8x10 8 、9x10 8 、1x10 9 、2x10 9 、3x10 9 、4x10 9 or 5x10 9 masked cardiomyocytes) can be administered to a subject with myocardial infarction (e.g., to replace dead or damaged cardiomyocytes).

[0305] Treatment of osteoarthritis and rheumatoid arthritis

[0306] In another example, the masked cells described herein can be used to treat osteoarthritis or rheumatoid arthritis. Osteoarthritis and rheumatoid arthritis (RA) are characterized by joint inflammation and are typically treated with anti-inflammatory therapeutic agents. To treat a subject with osteoarthritis or RA, masked cells can be modified to express anti-inflammatory biologics, such as TNFα inhibitors (e.g., TNFα inhibitory antibodies) that have been clinically used for the treatment of RA. Masked cells can be modified to produce anti-inflammatory biologics, such as TNFα inhibitors, by expressing a transgene encoding an anti-inflammatory biologic operably linked to a constitutive or inducible promoter. Masked cells (e.g., stem cells) expressing an anti-inflammatory biologic (e.g., TNFα inhibitor) can be differentiated into joint fibroblasts using methods known to those skilled in the art prior to administration to the joint, or isolated joint fibroblasts can be modified to express a masked transgene and an anti-inflammatory biologic. One million to one hundred million masked joint fibroblasts expressing an anti-inflammatory biologic (e.g., 1x10 6 、2x10 6 、3x10 6 、4x10 6 、5x10 6 、6x10 6 、7x10 6 、8x10 6 、9x10 6 、1x10 7 、2x10 7, 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 or 1x10 8 (masked articular fibroblasts) are injected into an arthritic or inflamed joint (depending on the size of the joint) to treat osteoarthritis or RA. Anti-inflammatory biologics that can be expressed by the masked 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 agents for treating RA (e.g., abatacept, rituximab).

[0307] Treatment of diabetes

[0308] Masked cells can be used to treat diabetes (e.g., type 1 or type 2 diabetes). Type 1 diabetes is caused by the pancreas being unable to produce enough insulin. Type 2 diabetes begins with insulin resistance but may develop insulin deficiency as the disease progresses. To treat a subject with diabetes, masked cells can be modified to express insulin, or cells that express insulin from a healthy subject (e.g., pancreatic beta cells from a subject without diabetes) can be modified to express one or more of the masked transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and SerpinB9 (Spi6) (e.g., one, two, three, four, five, six, seven, or all eight) and administered to the subject with diabetes. Masked cells can be modified to produce insulin by expressing a transgene encoding insulin operably linked to a constitutive or inducible promoter. The masked cells expressing insulin (e.g., stem cells) can be differentiated into insulin-producing cells (e.g., pancreatic beta cells) using methods known to those skilled in the art before administration or can be administered without differentiation, or isolated pancreatic beta cells can be modified to express the masked transgene and optionally a transgene encoding insulin. Eight hundred million to three billion masked pancreatic beta cells expressing insulin (e.g., expressing insulin endogenously or due to the expression of a transgene encoding insulin), such as 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 or 3x10 9(a masked pancreatic beta cell) is subcutaneously injected into a subject to generate a masked subcutaneous tissue that produces insulin for treating diabetes.

[0309] Treatment of hemophilia

[0310] In another example, the masked cells described herein can be used to treat hemophilia. Hemophilia patients do not produce functional factor VIII protein, which is a key blood component required for blood clotting. These patients can have severe bleeding, and the standard of care includes multiple injections of purified factor VIII protein per week. To treat a subject with hemophilia, masked cells can be modified to express an additional transgene encoding factor VIII. Factor VIII will be constitutively expressed in the masked cells by operably linking it to a constitutive promoter (such as CMV or CAG). Masked cells expressing factor VIII (such as stem cells) can be differentiated into cells that produce clotting factors (such as liver sinusoidal cells or endothelial cells) using methods known to those skilled in the art before administration or can be administered without differentiation, or isolated liver sinusoidal cells or endothelial cells expressing factor VIII from a healthy subject (e.g., a subject without hemophilia) can be modified to express one or more of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., one, two, three, four, five, six, seven, or all eight) and administered to a subject with hemophilia. If it is necessary to ensure a high level of expression of factor VIII, isolated liver sinusoidal cells or endothelial cells from a healthy subject that have been modified to express one or more masked transgenes can be further modified to express a transgene encoding factor VIII. Eight hundred million to three billion masked cells expressing factor VIII (e.g., endogenously expressing factor VIII or expressing factor VIII due to the expression of a transgene encoding factor VIII) (e.g., 8x10 8 、9x10 8 、1x10 9 、2x10 9 or 3x10 9 masked cells) are subcutaneously injected into a subject to generate a masked subcutaneous tissue that produces factor VIII for treating hemophilia.

[0311] Treatment of metabolic disorders

[0312] The masked cells of the present invention can also be used to treat inborn errors of metabolism. In most inborn errors of metabolism, the body does not produce a single enzyme, or produces it in a defective form. Inborn errors of metabolism include lysosomal storage diseases such as Hurler syndrome (α-L-iduronidase deficiency), Niemann-Pick disease (mutations in SMPD1, NPC1 or NPC2), Tay-Sachs disease (mutations in HEXA), Gaucher disease (mutations in the GBA gene), Fabry disease (α-galactosidase deficiency due to mutations in GLA) and Krabbe disease (galactosylceramidase deficiency due to mutations in GALC); galactosemia (galactokinase or galactose-1-phosphate uridyltransferase 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’s disease (glucose-6-phosphatase (G6PC) deficiency), GSD 2 / Pompe disease (acidic α-glucosidase (GAA) deficiency), GSD 3 / Cori’s disease or Forbes’disease (glycogen debranching enzyme AGL deficiency), GSD 4 / Anderson disease (glycogen branching enzyme (GBE1) deficiency), GSD 5 / McArdle disease (muscle glycogen phosphorylase (PYGM) deficiency), GSD 6 / Hers’disease (liver glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), GSD 7 / Tarui’s disease (muscle phosphofructokinase (PKFM) deficiency), GSD 9 (phosphorylase kinase (PHKA2, PHKB, PHKG2 or PHKA1) deficiency), GSD 10 (enolase 3 (ENO3) deficiency), GSD 11 (muscle lactate dehydrogenase (LDHA) deficiency), Fanconi-Bickel syndrome (glucose transporter 2 (GLUT2) deficiency), GSD 12 (aldolase A (ALDOA) deficiency), GSD 13 (β-enolase (ENO3) deficiency) or GSD 15 (glycogenin-1 (GYG1) deficiency);Mitochondrial disorders such as mitochondrial myopathy (Kearns-Sayre syndrome (KSS, caused by mitochondrial DNA deletions) and chronic progressive external ophthalmoplegia (CPEO, caused by mitochondrial DNA deletions or duplications or mutations in ANT1, POLG, POLG2 or PEO1), diabetes and deafness (DAD, caused by a mutation in mitochondrial DNA at position 3243 which encodes tRNALeu(UUR)), Leber hereditary optic neuropathy (LHON, caused by mutations in MT-ND1, MT-ND4, MT-ND4L and MT-ND6), Leigh syndrome (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, caused by a mutation in MT-ATP6), myoneurogenic gastrointestinal encephalopathy (MNGIE, caused by a mutation in TYMP), myoclonic epilepsy with ragged red fibers (MERRF, caused by mutations in MT-TK, MT-TL1, MT-TH, MT-TS1, MT-TS2 or MT-TF) or mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS, caused by mutations in MT-ND1, MT-ND5, MT-TH, MT-TL1 or MT-TV); Friedreich ataxia (mutation 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 (mutation in ABCD1); metal metabolism disorders such as Wilson disease (mutation in the Wilson disease protein ATP7B) and hemochromatosis (mutation in the human hemochromatosis protein HFE); organic acidemias 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 deficiency (ASL deficiency), argininosuccinate synthase 1 (ASS1) deficiency, citrin deficiency, carbamoyl phosphate synthase 1 (CPSI) deficiency, N-acetylglutamate synthase (NAGS) deficiency and ornithine translocase (ORNT1) deficiency.

[0313] For treating a subject having a metabolic disorder, masked cells can be modified to express the wild-type form of a gene that is mutated in the subject or a transgene encoding an enzyme that is absent or deficient in the subject (see Table 2), or cells from a healthy subject (e.g., a subject not having a metabolic disorder) that express the wild-type form of a gene that is mutated in the subject or an enzyme that is lacking 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 masking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject having a metabolic disorder. Expression of the wild-type form of a gene that is mutated in the subject or a transgene encoding an enzyme that is absent or deficient in the subject can be constitutively expressed in the masked cells by operably linking to a constitutive promoter (such as CMV or CAG) or can be inducibly expressed using one of the inducible expression systems described herein. Masked cells (such as stem cells) that are modified to express the wild-type form of a gene that is mutated in the subject or an enzyme that is absent or deficient in the subject can be differentiated into cells that normally express the gene or enzyme using methods known to those of skill in the art prior to administration or can be administered without differentiation, or isolated cells from a healthy subject that express the wild-type form of the gene or an enzyme that is mutated or lacking 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 masking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject having a metabolic disorder. If the subject has not been diagnosed as having a specific mutation prior to treatment, the subject can be evaluated using standard methods to identify the mutated gene associated with the metabolic disorder to ensure that the masked cells express the corresponding wild-type gene. Eight hundred million to three billion masked cells (e.g., 8x10 8 、9x10 8 、1x10 9 、2x10 9 or 3x10 9 masked cells) that express the wild-type form of a gene that is mutated in the subject can be subcutaneously injected to produce a masked subcutaneous tissue that produces the corresponding wild-type protein.

[0314] Methods of controlling the division of masked cells

[0315] In one aspect, a method of controlling cell proliferation at a transplantation site in an allogeneic host is provided (e.g., reducing the oncogenic potential of cells at the transplantation site or reducing the proliferation of cells that have become oncogenic at the transplantation site).

[0316] The method includes: providing a cell or population of such cells genetically modified to contain at least one mechanism for providing local immune containment at a transplantation site when transplanted into an allogeneic host; genetically modifying a cell division locus / plural cell division loci (CDL) in the cell, the CDL being one or more loci whose transcript is expressed by dividing cells (e.g., all dividing cells contain one or more of the immune containment transgenes), the genetic modification of the CDL including one or more of the following: a) an ablation linkage (ALINK) system that includes a DNA sequence encoding a negative selectable marker transcriptionally linked to the DNA sequence encoding the CDL; and b) a CDL-regulated inducible exogenous activator (EARC) system that includes an inducible activator-based gene expression system operably linked to the CDL; allowing proliferation of cells genetically modified to contain the ALINK system by maintaining the cells genetically modified to contain the ALINK system in the absence of an inducer of the negative selectable marker, and / or eliminating and / or inhibiting proliferation of cells genetically modified to contain the ALINK system by exposing the cells genetically modified to contain the ALINK system to an inducer of the negative selectable marker; and / or allowing proliferation of cells genetically modified to contain the EARC system by exposing the cells genetically modified to contain the EARC system to an inducer of the inducible activator-based gene expression system, or preventing or inhibiting proliferation of cells genetically modified to contain the EARC system by maintaining the cells genetically modified to contain the EARC system in the absence of an inducer of the inducible activator-based gene expression system; and transplanting the cell or cell population at a transplantation 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., 2, 3, 4 or more CDLs) can be referred to as "fail-safe cells". The fail-safe capacity is determined by the number of cells (e.g., clonal capacity) that the cells can grow before a single fail-safe cell loses the activity of all systems (e.g., ALINK or EARC) controlling cell division through genetic mutation (e.g., based on mathematical modeling, the number of cell divisions required for a cell to "escape" control and exhibit uncontrolled cell proliferation). The fail-safe capacity will depend on the number of ALINKs and the number of CDLs targeted by the ALINKs. The fail-safe properties are further described in Table 3.

[0317] Table 3. Using mathematical modeling to calculate the fail-safe cell capacity and its relationship to the human body. The model did not consider events in which the loss of CDL expression was concomitant with the loss of the activity of the negative selection marker, thereby impairing cell proliferation. Thus, the values were underestimated and assumed 10 of the negative selection marker 6 forward mutation rate was calculated. The estimated number of cells in the human body is 3.72x10 13 was taken from (Bianconi et al., 2013).

[0318]

[0319] In various embodiments, CDL is a locus identified as an "essential gene" as described in Wang et al., 2015 (which is incorporated herein by reference in its entirety). The essential genes in Wang et al., 2015 were identified by calculating a score (i.e., the CRISPR score) for each gene, which reflects the fitness cost imposed by the inactivation of the gene. In one embodiment, CDL has a CRISPR score (CS) of less than about -1.0 (Table 5, column 5).

[0320] In various embodiments, CDL is a locus / loci encoding a gene product related to cell division and / or replication (Table 5, column 6). For example, in various embodiments, CDL is a locus / loci encoding a gene product related to one or more of the following: i) the cell cycle; ii) DNA replication; iii) RNA transcription and / or protein translation; and iv) metabolism (Table 5, column 7).

[0321] In one embodiment, CDL is one or more cyclin-dependent kinases involved in regulating the processes of the cell cycle (e.g., controlling the G1 / S, G2 / M, and metaphase-to-anaphase transitions), such as CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, and / or CDK11 (Morgan, 2007). In one embodiment, CDL is one or more cyclins involved in controlling the cell cycle processes 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, CDL is one or more loci involved in the anaphase promoting complex, which controls the process of the metaphase-to-anaphase transition in the M phase of the cell cycle (Peters, 2002). In one embodiment, CDL is one or more loci involved in centromere components, which control the process of the metaphase-to-anaphase transition in the M phase of the cell cycle (Fukagawa, 2007). In one embodiment, CDL is one or more loci involved in microtubule components, which control the microtubule dynamics required for the cell cycle (Cassimeris, 1999).

[0322] In various embodiments, CDL is a locus / loci involved in housekeeping. As used herein, the term "housekeeping gene" or "housekeeping locus" refers to one or more genes required to maintain basic cellular functions. Housekeeping genes are expressed in all cells of an organism under normal and pathophysiological conditions.

[0323] In various embodiments, CDL is a locus / loci encoding a gene product related to cell division and / or proliferation and having a CRISPR score of less than about -1.0. For example, in one embodiment, CDL is a locus / loci encoding a gene product related to one or more of the following: i) the cell cycle; ii) DNA replication; iii) RNA transcription and / or protein translation; and iv) metabolism, and having a CRISPR score of less than about -1.0. In one embodiment, CDL can also be a housekeeping gene.

[0324] 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.

[0325] By linking the expression of the CDL to the expression of a DNA sequence encoding a negative selectable marker, cells can be modified into "fail-safe" cells, thereby allowing the elimination of mitotically active cells that express both the CDL and the negative selectable marker upon drug induction. For example, when cell proliferation is uncontrolled and / or accelerated relative to the normal division rate of the cells (such as the uncontrolled cell division exhibited by cancer cells), or when the therapeutic need for the cells has passed, it may be desirable to eliminate the proliferating cells. The elimination of proliferating cells can be achieved via genetic modification of the cells, referred to herein as "ablation linkage" (ALINK), which links the expression of a DNA sequence encoding a negative selectable marker to the expression of the CDL, thereby allowing the elimination or substantial inhibition of ALINK-modified proliferating cells and thus the expression of the CDL locus (substantial inhibition is inhibiting the cell proliferation rate to a rate too low to contribute to tumor formation). In the presence of a prodrug or other inducer of the negative selection system, cells expressing the negative selectable marker will stop proliferating or die, depending on the mechanism of action of the selectable marker. Cells can be modified to contain homozygous, heterozygous, hemizygous, or compound heterozygous ALINK. In one embodiment, to increase the fidelity of elimination, the negative selectable marker can be introduced into all functional alleles of the CDL. In a preferred embodiment, the negative selectable marker can be introduced into all functional alleles of the CDL. If desired, the fail-safe system can be used to eliminate all occult cells.

[0326] The ALINK can be inserted at any position of the CDL, which allows co-expression of the CDL and the negative selectable marker.

[0327] In some embodiments, the ALINK system includes the herpes simplex virus-thymidine kinase / ganciclovir system, the cytosine deaminase / 5-fluorocytosine system, the carboxylesterase / irinotecan system, or the iCasp9 / AP1903 system.

[0328] DNA encoding a negative selectable marker (e.g., HSV-TK) can be inserted into a CDL (e.g., CDK1) in a host cell such that expression of the negative selectable marker causes the host cell to express the negative selectable marker and, necessarily, causes the CDL to be killed in the presence of an inducer (e.g., a prodrug) of the negative selectable marker (e.g., ganciclovir (GCV)). In this example, a host cell modified with ALINK will produce thymidine kinase (TK), and the TK protein will convert GCV to GCV monophosphate, which is then converted to GCV triphosphate by cellular kinases. GCV triphosphate is incorporated into replicating DNA during the S phase, which results in termination of DNA elongation and apoptosis (Halloran and Fenton, 1998).

[0329] The modified HSV-TK gene (Preuß et al., 2010) is disclosed herein as an example of DNA encoding a negative selectable marker that can be used in ALINK genetic modification to selectively eliminate cells having an undesired cell division rate.

[0330] Alternative and / or additional negative selection systems are contemplated herein for use in the tools and / or methods provided herein. A variety of negative selectable marker systems are known in the art (e.g., dCK.DM (Neschadim et al., 2012)).

[0331] For example, in the field of “gene-directed enzyme / prodrug therapy” (GEPT), various clinically relevant negative selection systems have been actively developed with the aim of enhancing the therapeutic efficacy of conventional cancer therapies with little or no side effects (Hedley et al., 2007; Nawa et al., 2008). Generally, GEPT involves using viral vectors to deliver genes into cancer cells within or near cancer cell regions in mammalian cells where the enzyme that can convert a relatively non-toxic prodrug into a toxic agent is not found and produced.

[0332] HSV-TK / GCV, cytosine deaminase / 5-fluorocytosine (CD / 5-FC), and carboxylesterase / irinotecan (CE / CPT-11) are examples of negative selectable marker systems evaluated in GEPT preclinical and clinical trials (Danks et al., 2007; Shah, 2012).

[0333] To overcome the potential immunogenicity of the herpes simplex virus type 1 thymidine kinase / ganciclovir (TK / GCV) system, a "humanized" suicide system has been developed by engineering human deoxycytidine kinase to become thymidine-active and using it together with a non-toxic prodrug as a negative-selective (suicide) system antibody: bromovinyl-deoxyuridine (BVdU), L-deoxythymidine (LdT), or L-deoxyuridine (LdU) (Neschadim et al., 2012).

[0334] The CD / 5-FC negative-selective marker system is a widely used "suicide gene" system. Cytosine deaminase (CD) is a non-mammalian enzyme that can be obtained from bacteria or yeast (e.g., from Escherichia coli or Saccharomyces cerevisiae, respectively) (Ramnaraine et al., 2003). CD catalyzes the conversion of cytosine to uracil and is an important member of the pyrimidine salvage pathway in prokaryotes and fungi, but it 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 agent 5-FU, which has high levels of toxicity in humans (Ireton et al., 2002).

[0335] The CE / CPT-11 system is based on carboxylesterase, which is a serine esterase found in different tissues of mammalian species (Humerickhouse et al., 2000). The anticancer agent CPT-11 is a prodrug that is activated by CE to produce an active substance called 7-ethyl-10-hydroxycamptothecin (SN-38), which is 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).

[0336] Another example of a negative-selective marker system is the iCasp9 / AP1903 suicide system, which is based on modified human caspase 9 fused to the human FK506-binding protein (FKBP) to allow chemical dimerization using the small molecule AP1903, which has been safety tested in humans. Administration of the dimerizing drug induces apoptosis in cells expressing the engineered caspase 9 component. This system has several advantages, such as including low potential immunogenicity because it consists of human gene products, so the dimer drug only affects cells expressing the engineered caspase 9 component (Straathof et al., 2005). The iCasp / AP1903 suicide system is being tested in a clinical setting (Di Stasi et al., 2011).

[0337] In this article, it is considered that the negative selection marker system of the ALINK system can be replaced by a proliferation antagonist system. As used herein, the term "proliferation antagonist" refers to a natural or engineered compound whose presence inhibits (fully or partially) the division of cells. For example, Omomyc ER is a fusion protein of the MYC dominant negative Omomyc and a mutant murine estrogen receptor (ER) domain. When induced with tamoxifen (TAM), the fusion protein Omomyc ER localizes to the nucleus, where the dominant negative Omomyc dimerizes with C-Myc, L-Myc, and N-Myc, thereby sequestering them in a complex that cannot bind to the Myc DNA-binding consensus sequence (Soucek et al., 2002). Due to the lack of Myc activity, the cells cannot divide (Oricchio et al., 2014). Another example of a proliferation antagonist is A-Fos, which is a dominant negative activator protein-1 (AP1) (the heterodimer of the oncogene Fos and Jun) that inhibits DNA binding in equimolar competition (Olive et al., 1997). A-Fos can also be fused with the ER domain, thereby making its nuclear localization induced by TAM. Omomyc ER / tamoxifen or A-Fos ER / tamoxifen can be an alternative to TK / GCV to become ALINK.

[0338] By operably linking CDL to an EARC (such as an inducer-based gene expression system), cells can also be modified to be "fail-safe". Under these conditions, CDL will be expressed only in the presence of an inducer of the inducer-based gene expression system (and the cells can only divide). Under these conditions, depending on the mechanism of action of the inducer-based gene expression system and the function of CDL, in the absence of the inducer, EARC-modified cells stop dividing, slow down significantly, or die. Cells can be modified to contain homozygous or compound heterozygous EARC, or can be altered so that only the EARC-modified alleles can produce functional CDL. In one embodiment, for example, EARC modification can be introduced into all alleles of CDL to provide a mechanism for cell division control.

[0339] EARC can be inserted at any position of CDL that allows co-expression of CDL and the activator component of the inducible system in the presence of an inducer.

[0340] In one embodiment, an "activator"-based gene expression system is superior to a "repressor"-based gene expression system. For example, if a repressor is used to repress CDL, loss of repressor function mutations can release CDL expression, thereby enabling cell proliferation. In activation-based repression of cell division, loss of activator function (mutation) will turn off CDL expression, thereby preventing cell proliferation.

[0341] In some embodiments, the EARC system is a dox-bridge system, a coumarate switch-inducible system, an ecdysone-inducible system, a radio wave-inducible system, or a ligand-reversible dimerization system.

[0342] The dox-bridge can be inserted into 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. A host cell modified with a dox-bridge EARC can contain a reverse tetracycline transactivator (rtTA) gene (Urlinger et al., 2000) under the transcriptional control of a promoter, which is active in dividing cells (e.g., in CDL). This targeted insertion renders the CDL promoter no longer available for CDL transcription. To restore CDL transcription, a tetracycline-responsive element promoter (e.g., TRE (Agha-Mohammadi et al., 2004)) is inserted upstream of the CDL transcript, which will express the CDL gene only in the presence of expressed rtTA and doxycycline. When the only source of CDL expression is the dox-bridged allele, there is no CDL gene expression in the absence of doxycycline. The lack of CDL expression causes the EARC-modified cells to be impaired in their proliferation by death, cessation of cell division, or making the cell mitotic rate so slow that the EARC-modified cells cannot contribute to tumor formation.

[0343] As used herein, the term "dox-bridge" refers to a mechanism that separates the activity of a promoter from a target transcriptional region by expressing rtTA (Gossen et al., 1995) through an endogenous or exogenous promoter and enables transcription of the target region under the control of 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 consisting of TetO operator sequences upstream of a minimal promoter. After the binding of rtTA 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 into the same transcriptional unit as CDL or into different positions in the genome, as long as the permissive or non-permissive state of transcriptional expression of the target region is controlled by doxycycline. The dox-bridge is an example of EARC.

[0344] The present invention also contemplates introducing an EARC system into the 5' regulatory region of CDL.

[0345] The present invention contemplates that alternative and / or additional activator-based gene expression systems can be used in the tools and / or methods provided herein to generate EARC modifications. A variety of activator-based gene expression systems are known in the art.

[0346] For example, when cell division and / or proliferation is desired, a destabilizing protein domain (Banaszynski et al., 2006) fused to the effector protein product encoding CDL can be used in combination with a small molecule synthetic ligand to stabilize the CDL fusion protein. In the absence of the stabilizer, the unstable CDL protein will be degraded by the cell, which will then cease to proliferate. When the stabilizer compound is added, it will bind to the unstable CDL protein, which will not be degraded, thereby allowing cell proliferation.

[0347] For example, transcription activator-like effector (TALE) technology (Maeder et al., 2013) can be combined with dimerization-regulated expression induction (Pollock and Clackson, 2002). TALE technology can be used to generate a DNA-binding domain designed to be sequence-specific and placed together with a minimal promoter that replaces the CDL promoter. The TALE DNA-binding domain is also extended with a drug dimerization domain. The latter can bind to another engineered protein having a corresponding dimerization domain and a transcriptional activation domain.

[0348] For example, reverse-coumarate trans-activator (rcTA) can be inserted into the 5' untranslated region of CDL such that it will be expressed from the endogenous CDL promoter. Six repeats of the coumarate operator (6xCuO) can be inserted just before the translation start (ATG) of CDL. In the absence of coumarate in the system, rcTA cannot bind to 6xCuO, and thus CDL will not be transcribed because 6xCuO is inactive. When coumarate is added, it will form a complex with rcTA, thereby enabling binding to 6xCuO and enabling CDL transcription (Mullick et al., 2006).

[0349] For example, the N-terminal truncations of retinoid X receptor (RXR) and ecdysone receptor (EcR) fused to the activation domain of Vp16 (VpEcR) can be inserted into the 5' untranslated region of CDL such that they are co-expressed by the endogenous CDL promoter. An ecdysone response element (EcRE) with a downstream minimal promoter can also be inserted into CDL just upstream of the start codon. The co-expressed RXR and VpEcR can heterodimerize with each other. In the absence of ecdysone or the synthetic drug analogue muristerone A, the dimerized RXR / VpEcR cannot bind to the EcRE, and thus CDL is not transcribed. In the presence of ecdysone or muristerone A, the dimerized RXR / VpEcR can bind to the EcRE such that CDL is transcribed (No et al., 1996).

[0350] For example, transient receptor potential vanilloid-1 (TRPV1) together with ferritin can be inserted into the 5' untranslated region of CDL and co-expressed by the endogenous CDL promoter. A promoter that can be induced by NFAT (NFATre) can also be inserted into CDL just upstream of the start codon. Under normal circumstances, the NFAT promoter is inactive. However, after exposure to low-frequency radio waves, TRPV1 and ferritin generate Ca ++ waves that enter the cell, which in turn convert cytoplasmic NFAT (NFATc) into nuclear NFAT (NFATn), which ultimately will or ahu NFATre and transcribe CDL (Stanley et al., 2015).

[0351] For example, CDL can be functionally divided into multiple parts / domains: 5'-CDL and 3'CDL, and the FKBP peptide sequence can be inserted into each domain. An IRES (internal ribosome entry site) sequence can be placed between the two domains, which will be co-transcribed by the CDL promoter but will produce two separate proteins. In the absence of an inducer, the two separate CDL domains will be functionally inactivated. After introducing a dimerizing agent such as rapamycin or AP20187, the FKBP peptides will dimerize, thereby bringing the 5' and 3' CDL portions together and reconstructing the active protein (Rollins et al., 2000).

[0352] In one embodiment of the method, the genetically modified cell comprises: a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane-bound or locally acting and whose function is to attenuate the function of the host immune system (e.g., activation of white blood cells and NK cells that attack the graft) or to act as a defense mechanism against attacking white blood cells.

[0353] When transplanting cells or such cell populations into an allogeneic host, methods for genetically modifying the cells to include at least one mechanism for providing local immune containment at the transplant site are described, for example, in WO 2016 / 141480, the entire teachings of which are incorporated herein by reference.

[0354] The group of transgenes includes one or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6). In one embodiment, the group of transgenes includes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).

[0355] Optionally, the method further includes expressing in the cells one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39. In one embodiment, the TGF-β or the biological agent acts locally in the graft environment.

[0356] Techniques for introducing various genetic modifications, such as transgenes, into animal cells are described herein and are generally known in the art.

[0357] In one embodiment of the method, the cells are stem cells, cells suitable for genome editing, or cells that can serve as a source of a therapeutic cell type (e.g., cells that can be directed to differentiate into lineage-restricted or terminally differentiated cells useful for cell therapy, or cells of the desired target tissue). In one embodiment, 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 progenitors, germline stem cells, lung stem cells or progenitors, 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 progenitors. In some embodiments, the cells are derived from the 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, such as mammalian cells, such as human or mouse cells.

[0358] Techniques for transplanting genetically modified cells into the transplant site of an allogeneic host are described herein and are generally known in the art.

[0359] In various embodiments of any of the methods of the present disclosure, the host has a degenerative disease or disorder treatable by cell therapy. Examples of such diseases or disorders include, but are not limited to: blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, lung diseases, blood diseases, neurological diseases (such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS), enzyme or hormone deficiencies, metabolic disorders (e.g., lysosomal storage diseases, galactosemia, maple syrup urine disease, phenylketonuria, glycogen storage diseases, mitochondrial disorders, Friedrich's ataxia, peroxisomal disorders, metal metabolism disorders, or organic acidemias), autoimmune diseases (e.g., psoriasis, systemic lupus erythematosus, Graves' disease, inflammatory bowel disease, Addison's disease, Sjogren's syndrome), Hashimoto's thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinitis pigmentosa, 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, keratoglobus, muscular dystrophy, neuronal ceroid lipofuscinosis, previous diseases, progressive supranuclear palsy, Sandhoff disease, spinal muscular atrophy, retinitis pigmentosa) or age-related diseases (e.g., atherosclerosis, cardiovascular diseases (such as angina pectoris, myocardial infarction), cataracts, osteoporosis, or hypertension).

[0360] Drug composition

[0361] The masked cells described herein can be incorporated into a vehicle for administration to a patient, such as a human patient receiving a graft or having a disease or disorder described herein. Methods known in the art can be used to prepare a drug composition containing the masked cells. For example, such compositions can be prepared using, for example, physiologically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacology, 22nd Edition, Allen, L. Ed. (2013); which is incorporated herein by reference) and in the desired form, such as in the form of an aqueous solution.

[0362] The masked cells described herein can be administered in any physiologically compatible carrier such as a buffered saline solution. Pharmaceutically acceptable carriers and diluents include saline, buffered aqueous solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art. Other examples include liquid 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 glycols, and mixtures thereof, as well as in oils. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, etc.), suitable mixtures thereof, and / or vegetable oils. For example, by using a coating such as lecithin, in the case of a dispersion, by maintaining the desired particle size and by using surfactants to maintain proper fluidity. In many cases, it is preferred to include isotonic agents such as sugars or sodium chloride. The solution is preferably sterile and has a fluidity to the extent that it is easy to inject. Preferably, the solution is stable under the manufacturing and storage conditions and is preserved against the contaminating action of microorganisms such as bacteria and fungi by using, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. The solution of the present invention can be prepared by using a pharmaceutically acceptable carrier or diluent and other ingredients listed above as needed, followed by filtration sterilization and then incorporation of the masked cells as described herein.

[0363] For example, if desired, the solution containing the pharmaceutical composition described herein can be appropriately buffered and first made isotonic with a sufficient amount of saline or dextrose in the liquid diluent. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art from the present disclosure. The individual responsible for administration will determine the appropriate dose for the individual subject in any case. In addition, for human administration, the formulation can meet the requirements of the FDA Office for the standards of sterility, pyrogenicity, general safety, and purity of biological products.

[0364] A pharmaceutical composition comprising a masked cell in a semi-solid or solid carrier is typically formulated for surgical implantation at a transplantation site or an affected site of a disease or disorder in a subject. It should be understood that liquid compositions can also be administered by surgical procedures. In certain embodiments, the semi-solid or solid pharmaceutical composition can comprise a semi-permeable gel, matrix, cell scaffold, etc., which can be non-biodegradable or biodegradable. For example, in some embodiments, it may be desirable or appropriate to isolate the masked cells from their surroundings, but still enable the cells to secrete biomolecules and deliver the biomolecules (e.g., the therapeutic agents listed in Table 2) to the surrounding cells.

[0365] In other embodiments, different types of degradable gels and networks are used in the pharmaceutical compositions of the present invention. For example, degradable materials include biocompatible polymers such as poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, etc.

[0366] In another embodiment, one or more hydrogels are used in the pharmaceutical composition. One or more hydrogels can include collagen, telopeptide-free collagen, fibrin constructs, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, and poly(ethylene oxide). In addition, hydrogels can be formed from poly(2-hydroxyethyl methacrylate), poly(acrylic acid), self-assembling peptides (e.g., RAD16), poly(methacrylic acid), poly(N-vinyl-2-pyrrolidone), poly(vinyl alcohol), and their copolymers with each other and with hydrophobic monomers such as methyl methacrylate, vinyl acetate, etc. Also preferred are hydrophilic polyurethanes containing large poly(ethylene oxide) blocks. Other preferred materials include hydrogels comprising interpenetrating networks of polymers that can be formed by addition or by condensation polymerization, the components of which can comprise hydrophilic and hydrophobic monomers such as those just listed. In-situ formed degradable networks are also suitable for the present invention (see, for example, Anseth, K S et al. J. Controlled Release, 2002; 78:199-209; Wang, D. et al., Biomaterials, 2003; 24:3969-3980; U.S. Patent Publication 2002 / 0022676). These in-situ formed materials are formulated as fluids suitable for injection; the hydrogels can then be induced to form by various means such as changes in temperature, pH, and in-situ or in-vivo exposure to light. In one embodiment, the construct comprises a gel containing fibrin glue. In another embodiment, the construct comprises a gel containing telopeptide-free collagen.

[0367] The polymers used to form the matrix can be in the form of hydrogels. Generally, a hydrogel is a cross-linked polymeric material that can absorb more than 20% of its weight in water while maintaining a unique three-dimensional structure. This definition includes dry cross-linked polymers that will swell in an aqueous environment as well as water-swellable materials. Many hydrophilic polymers can be cross-linked to produce hydrogels, whether the polymers are of biological origin, semi-synthetic, or fully synthetic. Hydrogels can be produced from synthetic polymeric materials. Such synthetic polymers can be customized to have a range of properties and predictable batch-to-batch consistency and represent a reliable source of material that generally does not have to be concerned about immunogenicity. The matrix can include hydrogels formed from self-assembling peptides, such as those discussed in U.S. Patent Nos. 5,670,483 and 5,955,343, U.S. Patent Application No. 2002 / 0160471, and PCT Application No. WO 02 / 062969.

[0368] Properties that make hydrogels valuable in drug delivery applications include equilibrium swelling, adsorption kinetics, solute permeability, and their in vivo performance characteristics. The permeability of a compound depends in part on the degree of swelling or water content and the rate of biodegradation. Since the mechanical strength of the gel may decrease proportionally with the degree of swelling, it is also within the contemplation of the present invention that the hydrogel can be attached to a substrate such that the composite system enhances the mechanical strength. In some embodiments, the hydrogel can be impregnated in a porous substrate in order to obtain the mechanical strength of the substrate as well as the useful delivery properties of the hydrogel.

[0369] In other embodiments, the pharmaceutical composition comprises a biocompatible matrix made of natural, modified natural, or synthetic biodegradable polymers (including homopolymers, copolymers, and block polymers and combinations thereof).

[0370] Examples of suitable biodegradable polymers or polymer classes include any biodegradable polymers discussed in the present disclosure, including but not limited to fibrin, collagens type I, II, III, IV, and V, elastin, gelatin, vitronectin, fibronectin, laminin, thrombin, poly(amino acids), oxidized cellulose, tropoelastin, filamentous fibrils, ribonucleic acid, deoxyribonucleic acid, proteins, polynucleotides, gum arabic, recombinant basement membrane matrix, starch, dextran, alginate, hyaluronic acid, chitin, chitosan, agarose, polysaccharides, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polyethylene glycol, acellular tissue, self-assembling peptides, polypeptides, glycosaminoglycans, their derivatives, and mixtures thereof. Suitable polymers also include poly(lactide) (PLA), which can be formed from L(+) and D(-) polymers, polyhydroxybutyrate, polyurethanes, polyphosphazenes, poly(ethylene glycol)-poly(lactide-co-glycolide) copolymers, degradable polycyanoacrylates, and degradable polyurethanes. 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.

[0371] 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), poly(fumarates), polydioxanone, polycarbonates, polyoxalates, poly(α-hydroxy acids), poly(esters), polyurethanes, poly(ester urethanes), poly(ether urethanes), polyanhydrides, polyacetates, polycaprolactone, poly(orthoesters), polyamino acids, polyamides, and blends and copolymers thereof. Additionally useful biodegradable polymers include but are not limited to stereopolymers of L- and D-lactic acid, copolymers of bis(p-carboxyphenoxy)propane and sebacic acid, sebacic acid copolymers, copolymers of caprolactone, poly(lactic acid) / poly(glycolic acid) / polyethylene glycol copolymers, copolymers of polyurethanes and poly(lactic acid), copolymers of α-amino acids, copolymers of α-amino acids and hexanoic acid, copolymers of α-benzyl glutamate and polyethylene glycol, copolymers of succinate esters and poly(diols), polyphosphazenes, poly(hydroxyalkanoates), and mixtures thereof. Binary and ternary systems are also contemplated.

[0372] Generally, the material used to form the matrix is desirably configured such that it: (1) has mechanical properties suitable for the intended application; (2) remains intact enough until tissue ingrowth and healing occur; (3) does not cause an inflammatory or toxic response; (4) is metabolized in the body after achieving its purpose; (5) is easily processed into the desired final product to be shaped; (6) exhibits an acceptable shelf life; and (7) is easily sterilized.

[0373] In another embodiment, a masked cell population can be administered by using a scaffold. The composition, shape, and porosity of the scaffold can be any of those described above. Generally, these three-dimensional biomaterials contain live cells attached to, dispersed within, or incorporated and embedded in the extracellular matrix of the scaffold. Once implanted into the target area of the body, these implants become integrated with the host tissue, where the transplanted cells gradually become established.

[0374] Non-limiting examples of scaffolds that can be used include fabric structures such as woven fabrics, knitted fabrics, braided fabrics, mesh fabrics, non-woven fabrics, and warp knitted fabrics; porous foams, semi-porous foams, perforated membranes or sheets, microparticles, beads, and spheres, and composite structures that are combinations of the above structures. Non-woven pads can be formed, for example, using fibers composed of a synthetic absorbable copolymer of glycolic acid and lactic acid (PGA / PLA), which are sold under the trade name VICRYL suture (Ethicon, Inc., Somerville, N.J.). Foams composed of, for example, poly(ε-caprolactone) / poly(glycolic acid) (PCL / PGA) copolymers and formed by methods such as freeze-drying or lyophilization, as discussed in U.S. Patent No. 6,355,699, can also be utilized.

[0375] In another embodiment, the framework is a felt, which can be composed of multifilaments made from a bioabsorbable material. Standard fabric processing techniques including crimping, cutting, carding, and needling can be used to make the yarns into felt. In another embodiment, cells are seeded onto a foam scaffold that can be used as a composite structure.

[0376] The framework can be molded into useful shapes to fill tissue voids. Thus, the framework can be shaped to provide not only a channel for nerve growth but also a scaffold for supporting and surrounding tissues such as vascular tissue, muscle tissue, etc. In addition, it will be understood that a cell population can be cultured on a preformed, non-degradable surgical or implantable device.

[0377] The pharmaceutical composition may comprise a preparation made from shielded cells, which are formulated together with a pharmaceutically acceptable carrier or vehicle. Suitable pharmaceutically acceptable carriers include any of the carriers discussed in the present disclosure, including but not limited to water, saline solutions (such as Ringer's solution), alcohols, oils, gelatin, polyvinylpyrrolidone, carbohydrates (such as lactose, amylose or starch), fatty acid esters, and hydroxymethylcellulose. Such preparations may be sterilized and, if desired, may be mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifying agents, salts affecting osmotic pressure, buffers, and coloring agents. Pharmaceutical carriers suitable for the present invention are known in the art and are described, for example, in Pharmaceutical Sciences (17th Edition, Mack Pub. Co., Easton, Pa.) and WO 96 / 05309.

[0378] Method of treatment

[0379] The shielded cells and compositions described herein may be administered to a subject in need thereof (e.g., a subject who is receiving or has received a graft, or a subject suffering from a disease or disorder described herein) by a variety of routes, such as topical administration to or near the graft site, topical administration to a site affected by a disease or disorder (e.g., injection into a joint for the treatment of RA, injection into the subretinal space for the treatment of wet AMD, direct administration to the central nervous system (CNS) (e.g., intracerebral, intraventricular, intrathecal, intracisternal or stereotactic administration) for the treatment of neurological diseases such as Parkinson's disease, direct injection into the myocardium for the treatment of myocardial infarction), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration. The most suitable route of administration in any given case will depend on the particular cells or composition being administered, the patient, the method of drug formulation, the method of administration (e.g., time of administration and route of administration), the age, weight, sex of the patient, the severity of the disease being treated, the diet of the patient, and the excretion rate of the patient. 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 topical administration, the shielded cells may be administered by any means of placing the cell population in the desired location, including catheters, syringes, shunts, stents, microcatheters, pumps, implantation with a device or implantation with a stent.

[0380] As described herein, prior to administration, the cell population can be incubated in the presence of one or more factors or under conditions that stimulate the stem cells to differentiate into the desired cell type (e.g., neurons, cardiomyocytes, RPE cells, insulin-producing cells, clotting factor-producing cells, articular fibroblasts, or other cell types described herein). Such factors are known in the art, and those skilled in the art will understand that suitable differentiation conditions can be determined by routine experimentation. Such factors include growth or nutritional factors, chemokines, cytokines, cell products, demethylating agents, and other stimulants known to stimulate the differentiation of stem cells, for example, along angiogenic, hemangiogenic, vasculogenic, skeletal muscle, vascular smooth muscle, pericytes, neuronal, or vascular endothelial pathways or lineages. Alternatively, the composition administered to the patient comprises a masked cell population having one or more factors that stimulate cell differentiation into the desired cell type, wherein cell differentiation occurs in vivo at the tissue site. In some embodiments, the masked 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.

[0381] In some embodiments, cells of a particular cell type are collected from a patient or from a donor (e.g., from an HLA-matched or mismatched donor, such as a donor without a disease or disorder), modified to express one or more (e.g., one, two, three, four, five, six, seven, or eight) masked transgenes, and then administered to a subject. This method can be used to treat a subject carrying a mutation in a particular gene, because the masked donor cells can endogenously express the wild-type form of the gene; or to a subject lacking a particular secreted protein or enzyme (e.g., using masked donor cells that endogenously express the protein or enzyme lacking in the subject). This method can also be used to treat a subject receiving an organ or tissue transplant, 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) masked transgenes prior to transplantation.

[0382] Subjects treatable as described herein are subjects who have received a graft, or subjects having a disease or disorder described herein (e.g., wet AMD or retinal dystrophy, neurodegenerative diseases such as Parkinson's disease, heart infarction, osteoarthritis or RA, diabetes, hemophilia, metabolic disorders or diseases or disorders listed in Table 2). The cells, compositions and methods described herein can be used to treat diseases or disorders caused by or associated with cell loss, mutations or deficiencies of proteins or abnormal production of proteins, which can be treated using cell replacement proteins or cell therapies, production of therapeutic proteins, production of agonist antibodies or production of inhibitory antibodies. The methods described herein can include the step of screening a subject for mutations in genes associated with insufficient protein production prior to treating or administering the compositions described herein to the subject. Standard methods known to those of skill in the art (e.g., genetic testing) can be used to screen the subject for genetic mutations. The methods described herein can also include the step of evaluating the symptoms of the disease or disorder of the subject prior to treating or administering the masked cells or compositions described herein to the subject. After administration of the masked cells or compositions, the same diagnostic tests can be used to evaluate the subject to determine if the subject's condition has improved. The compositions and methods described herein can be administered as a prophylactic treatment to a patient who has received a tissue or organ graft prior to the patient showing any signs of tissue or organ rejection.

[0383] The masked cells, compositions and methods described herein can be used to replace dead or dying cells in a subject (e.g., replace neurons in a subject having a neurodegenerative disease, or replace cardiomyocytes in a subject having a myocardial infarction). The masked cells, compositions and methods described herein can also be used to provide immune containment in the area of a tissue or organ graft, or reduce the risk of tissue or organ graft rejection. Masked cells expressing a therapeutic agent (such as a protein or agonist antibody), compositions comprising such cells, or methods of administering such cells can be used to replace or provide the wild-type form of a protein that is mutated or deficient in a subject (e.g., a protein that is produced but cannot function properly due to a genetic mutation, such as a truncated protein or a protein with an altered charge, polarity or binding pattern; or a protein that is not produced or is produced in insufficient amounts, such as an insufficient protein production associated with the diseases or disorders in Table 2). Masked cells expressing a therapeutic agent (such as an inhibitory or neutralizing antibody), compositions comprising such cells, or methods of administering such cells can be used to block or neutralize a protein that is overexpressed or abnormally produced in a subject (e.g., a protein that is produced at a different time or location than the production of the protein in a healthy subject, such as an abnormal protein production associated with the diseases or disorders listed in Table 2).

[0384] Treatment may include administering masked cells or a composition containing various unit doses of masked cells. Each unit dose will typically contain a predetermined amount of the masked cells described herein. The amount to be administered and the specific route of administration and formulation are within the skill of the person of ordinary skill in the clinical art. The unit dose need not be administered as a single injection, but may include a continuous infusion over a set period of time. Administration can be effected using a catheter, syringe, shunt, stent, microcatheter, pump, implantation with a device or implantation with a stent. The number of cells administered can vary depending on whether the cells are to be administered to a tissue, organ or body part associated with a disease or injury or subcutaneously to create a masked subcutaneous tissue. For administration to a tissue, organ or body part, the masked cells can be, for example, from 1x10 4 cells to 1x10 10 cells (e.g., 1x10 4 , 2x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 , 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x108 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 cells) are administered to the patient. The number of cells administered will depend on the size of the recipient tissue, organ, or body part. For example, 2.5x10 4 to 1x10 5 cells (e.g., 2.5x10 4 , 3x10 4 , 4x10 4 , 5x10 4 , 6x10 4 , 7x10 4 , 8x10 4 , 9x10 4 or 1x10 5 cells) may be administered (e.g., injected) into the subretinal space of the eye or a specific brain region; 1x10 6 to 1x10 8 cells (e.g., 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 or 1x10 8 cells) may be administered (e.g., injected) into a joint, where the amount of cells depends on the size of the joint; and 5x10 8from 5x10 9 to 5x10 8 cells (e.g., 5x10 8 cells, 6x10 8 cells, 7x10 8 cells, 8x10 8 cells, 9x10 9 cells, 1x10 9 cells, 2x10 9 cells, 3x10 9 cells, 4x10 9 cells or 5x10 8 cells) may be administered to the myocardium. To create a shielded subcutaneous tissue, 8x10 9 cells to 3x10 8 cells (e.g., 8x10 8 cells, 9x10 9 cells, 1x10 9 cells, 2x10 9 cells) may be administered subcutaneously (e.g., by injection). The shielded cells may be administered in two or more doses (e.g., two, three, four, five or more different doses, e.g., to different sized joints of a patient with RA) or the same dose may be administered two or more times (e.g., two, three, four, five, six or more times over the course of an hour, a day, a week, a month or a year). In some embodiments, the shielded cells described herein are administered in the form of tissue (e.g., tissue that has been grown and / or differentiated in vitro from the shielded cells). In some embodiments, the shielded tissue is administered (e.g., implanted) with a gel, biocompatible matrix or scaffold.

[0385] The compositions described herein are administered in an amount sufficient to prevent or reduce transplant rejection or to ameliorate the symptoms of the diseases or disorders listed in Table 2 (e.g., to reduce the symptoms of osteoarthritis or RA (e.g., to reduce inflammation, joint pain, stiffness or immobility); to reduce the symptoms of retinitis pigmentosa or wet AMD (e.g., to improve vision, slow or reduce angiogenesis in the eye); to reduce the symptoms of Parkinson's disease (e.g., to reduce tremors, rigidity, bradykinesia or to improve posture or gait); to reduce the symptoms of diabetes (e.g., to increase insulin levels, reduce the need for regular insulin injections); to reduce the symptoms of myocardial infarction (e.g., to improve cardiac function, reduce infarct size); to reduce the symptoms of hemophilia (e.g., to increase the levels of clotting factors such as factor VIII, reduce excessive bleeding, reduce bruising, reduce nosebleeds, reduce joint pain or swelling); or to reduce the symptoms of a metabolic disorder (e.g., increased appetite, growth or weight gain or to reduce lethargy, weight loss, jaundice, seizures, abdominal pain or vomiting)). Transplant rejection can be evaluated using standard methods known to those of skill 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 the transplant rejection rate typically observed without treatment. In some embodiments, administration of the masked cells or compositions described herein results in an outcome equivalent to that observed in subjects administered an immunosuppressive agent for transplant rejection. The symptoms of the diseases and disorders described herein can be evaluated using standard methods known to those of skill in the art and can be reduced (e.g., the condition of the subject can be improved) by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) compared to the symptoms prior to administration of the masked cells or compositions described herein. These effects can occur, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks or longer after administration of the compositions described herein. The patient can be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer after administration of the masked cells or compositions, depending on the dose and route of administration used for treatment. Based on the evaluation results, the patient can receive additional treatment.

[0386] Combination therapy

[0387] In some embodiments, the masked cells described herein are administered in combination with one or more additional therapeutic agents. The additional therapeutic agent(s) may be administered before, after, or simultaneously with the administration of the masked cells. The masked cells and the additional therapeutic agent(s) may also be administered simultaneously via co-formulation. The masked cells and the therapeutic agent(s) may also be administered sequentially such that the actions of the masked cells and the therapeutic agent(s) overlap and their combined action is such that the reduction in symptoms or other parameters associated with the disorder is greater than the reduction observed with the masked cells or the therapeutic agent(s) delivered alone or in the absence of the other. The actions of the masked cells and the therapeutic agent(s) may be partially additive, fully additive, or more than additive (e.g., synergistic). Sequential or substantially simultaneous administration of the masked cells and the therapeutic agent(s) may be achieved by any suitable route, including but not limited to oral, intravenous, intramuscular, topical, or subcutaneous routes. The masked cells and the therapeutic agent(s) may be administered by the same route or different routes. For example, the masked cells may be administered by subcutaneous injection while the additional therapeutic agent is administered orally. The masked cells may be administered immediately before or 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, 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or up to 1 - 7, 1 - 14, 1 - 21, or 1 - 30 days.

[0388] In one instance, the additional therapeutic agent is one or more immunosuppressive agents that are commonly administered for organ or tissue transplantation. The immunosuppressive agent can be an agent that is administered immediately after transplantation to prevent acute rejection (e.g., methylprednisolone, atgam, antithymocyte globulin, OKT3, basiliximab, or daclizumab) or an immunosuppressive agent for maintenance (e.g., prednisone), a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), mycophenolate mofetil, azathioprine, or sirolimus). Other immunosuppressive agents administered after an organ graft include corticosteroids (e.g., methylprednisolone, dexamethasone, prednisolone), cytotoxic immunosuppressive agents (e.g., azathioprine, chlorambucil, cyclophosphamide, mercaptopurine, methotrexate), immunosuppressive agent antibodies (e.g., antithymocyte globulin, basiliximab, infliximab), sirolimus derivatives (e.g., everolimus, sirolimus), and antiproliferatives (e.g., mycophenolate mofetil, sodium mycophenolate, and azathioprine). In such a case, the masked cells are administered to the transplantation site or near the transplantation site, or the tissue to be transplanted is modified to express one or more (e.g., one, two, three, four, five, six, seven, or eight) masked transgenes, and the immunosuppressive agent is administered as another source of immune suppression when needed.

[0389] For use in treating inflammatory and autoimmune-related diseases or disorders, additional agents can be disease-modifying anti-rheumatic drugs (DMARDs), biologic response modifiers (a type of DMARD), corticosteroids, or non-steroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the additional agent is prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, or a biologic such as tofacitinib, adalimumab, abatacept, anakinra, anakinra, certolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab. In some embodiments, the additional agent is 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab (Lemtrada), aminosalicylates (5-aminosalicylic acid, sulfasalazine, mesalamine, balsalazide, olsalazine), antibiotics, antihistamines, anti-TNFα (infliximab, adalimumab, certolizumab pegol, natalizumab), azathioprine, belimumab, beta interferon, calcineurin inhibitors, certolizumab, corticosteroids, cromolyn, cyclosporine A, cyclosporine, dimethyl fumarate (tecfidera), etanercept, fingolimod (Gilenya), fumarates, glatiramer acetate (Copaxone), golimumab, hydroxyurea, IFNγ, IL-11, infliximab, leflunomide, leukotriene receptor antagonists, long-acting beta2 agonists, mitoxantrone, mycophenolate mofetil, natalizumab (tysabri), ocrelizumab, pimecrolimus, probiotics (VSL#3), retinoids, rituximab, salicylates, short-acting beta2 agonists, sulfasalazine, tacrolimus, teriflunomide (Aubagio), theophylline, tocilizumab, ustekinumab (anti-IL-12 / IL-23), or vedolizumab (anti-α4β7 integrin). In such cases, masked cells can be administered to replace tissues or organs damaged by inflammatory or autoimmune-related diseases or disorders. In another example, the administered masked cells can be modified to express a biotherapeutic agent (e.g., an antibody) for treating a specific inflammatory or autoimmune-related disease or disorder, and the additional agent can be a compound or a general anti-inflammatory agent (e.g., an NSAID or a corticosteroid).

[0390] For example, if the disease is rheumatoid arthritis, the additional agent(s) can be one or more of the following: prednisone, prednisolone, and methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, and azathioprine, tofacitinib, adalimumab, abatacept, anakinra, anakinra, certolizumab, etanercept, golimumab, infliximab, rituximab, or tocilizumab. The sheltered cells administered can be cartilage or bone-producing cells of a joint. In some embodiments, the sheltered cells can be modified to produce an anti-TNFα antibody and can be administered in combination with an anti-inflammatory agent (such as a corticosteroid).

[0391] In another example, for treating AMD or a retinal dystrophy, the additional therapeutic agent can be an additional biologic agent (e.g., bevacizumab, ranibizumab, or aflibercept), photodynamic therapy, or photocoagulation. The sheltered cells administered can be retinal cells (e.g., RPE cells). In some embodiments, the sheltered cells can be modified to produce a VEGF inhibitor and can be administered in combination with photodynamic therapy or photocoagulation.

[0392] For treating Parkinson's disease, the sheltered cells described herein can be administered with carbidopa-levodopa, dopamine agonists (e.g., pramipexole, ropinirole, rotigotine, or apomorphine), MAO-B inhibitors (e.g., selegiline, rasagiline), catechol-O-methyltransferase inhibitors (e.g., entacapone or tolcapone), anticholinergics (e.g., benztropine or trihexyphenidyl), amantadine, or deep brain stimulation. The sheltered cells administered can be dopaminergic neurons.

[0393] Additional agents for treating myocardial infarction include anticoagulants (e.g., rivaroxaban, dabigatran, apixaban, heparin, warfarin), antiplatelet agents (e.g., aspirin, clopidogrel, dipyridamole, prasugrel, ticagrelor), angiotensin-converting enzyme inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril), angiotensin II receptor blockers (e.g., candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan), angiotensin receptor neprilysin inhibitors (e.g., sacubitril / valsartan), β-blockers (e.g., acebutolol, atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, sotalol), combined α and β-blockers (e.g., carvedilol, labetalol hydrochloride), calcium channel blockers (e.g., amlodipine, diltiazem, felodipine, nifedipine, nimodipine, nisoldipine, verapamil), cholesterol-lowering drugs (e.g., statins (e.g., atorvastatin, rosuvastatin), niacin (e.g., lovastatin), cholesterol absorption inhibitors (e.g., ezetimibe / simvastatin)), digitalis preparations (e.g., lanoxin), diuretics (e.g., amiloride, bumetanide, chlorothiazide, chlorthalidone, furosemide, hydrochlorothiazide, indapamide, spironolactone), vasodilators (e.g., isosorbide dinitrate, nesiritide, hydralazine, nitrates, minoxidil), dual antiplatelet therapy (e.g., aspirin and a P2Y12 inhibitor) or cardiac surgery (e.g., angioplasty, artificial heart valve surgery, atherectomy, bypass surgery, cardiomyoplasty, heart transplantation, minimally invasive cardiac surgery, radiofrequency ablation, stent surgery or myocardial revascularization). The administered masked cells can be cardiomyocytes.

[0394] For use in treating infectious diseases, additional agents can be antiviral compounds (e.g., vidarabine, acyclovir, ganciclovir, valganciclovir, nucleoside analogue reverse transcriptase inhibitors (NRTIs) (e.g., AZT (zidovudine), ddI (didanosine), ddC (zalcitabine), d4T (stavudine) or 3TC (lamivudine)), non-nucleoside reverse transcriptase inhibitors (NNRTIs) (e.g., (nevirapine or delavirdine), protease inhibitors (saquinavir, ritonavir, indinavir or nelfinavir), ribavirin or interferon); antibacterial compounds; antifungal compounds; antiparasitic compounds. The administered masked cells can be immune cells (e.g., cells that can help fight infectious diseases, such as masked T cells or B cells).

[0395] For use in the treatment of diabetes, additional agents can be insulin, sulfonylureas (e.g., chlorpropamide, glipizide, glyburide, glimepiride), biguanides (e.g., metformin), meglitinides (e.g., repaglinide, nateglinide), thiazolidinediones (e.g., rosiglitazone, pioglitazone), DPP-4 inhibitors (sitagliptin, saxagliptin, linagliptin, alogliptin), SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin), α-glucosidase inhibitors (e.g., acarbose, miglitol), bile acid sequestrants (e.g., colesevelam), aspirin or a dietary regimen. The masked cells administered can be pancreatic β cells, which can optionally be modified to express a transgene encoding insulin.

[0396] For use in the treatment of hemophilia, additional therapeutic agents can be clotting factors, desmopressin, clot-preserving drugs (e.g., antifibrinolytics such as aprotinin, aminocaproic acid, fibrinogen or tranexamic acid), fibrin sealants or physical therapy. The masked cells administered can be liver sinusoidal cells or endothelial cells, which can optionally be modified to express a transgene encoding factor VIII.

[0397] For the treatment of metabolic deficiencies or disorders, additional therapeutic agents can be coenzymes (e.g., biotin, hydroxocobalamin, riboflavin, pyridoxine, folic acid, thiamine, ubiquinone, tetrahydrobiopterin), bone marrow grafts, organ grafts (e.g., liver, kidney or heart transplants), hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium-chain triglycerides, miglustat, enzyme replacement therapy or dietary restriction (e.g., for patients with phenylketonuria, a low-protein or phenylalanine-restricted diet). The masked cells can be cells carrying a wild-type copy of a gene mutated in a subject with a metabolic disorder, or cells that endogenously produce an enzyme that is lacking in a subject with a metabolic disorder (e.g., hepatocytes, renal cells, cardiac cells or any other cells carrying a wild-type copy of a gene mutated or producing an enzyme that is lacking in a subject with a metabolic disorder).

[0398] For use in the treatment of cancer, additional agents can be checkpoint inhibitors, chemotherapeutic drugs, biopharmaceuticals, non-pharmacological therapies (e.g., radiotherapy, cryotherapy, hyperthermia or surgical resection of tumor tissue) or anti-cancer vaccines. The masked cells administered can be immune cells that can help fight cancer (e.g., macrophages, natural killer cells, dendritic cells or T cells).

[0399] Checkpoint inhibitors can be classified into at least four major categories: i) agents that directly block inhibitory pathways on T cells or natural killer (NK) cells such as antibodies (e.g., antibodies targeting PD-1 such as nivolumab, pidilizumab / CT-011, and pembrolizumab, antibodies targeting TIM-3, and antibodies targeting LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, or KIR), ii) agents that directly activate stimulatory pathways on T cells or NK cells such as antibodies (e.g., antibodies targeting OX40, GITR, or 4-1BB), iii) agents that block inhibitory pathways on immune cells or rely on antibody-dependent cytotoxicity to ablate inhibitory populations of immune cells such as antibodies (e.g., antibodies targeting CTLA-4 such as ipilimumab or tremelimumab), antibodies targeting VISTA, and antibodies targeting PD-L2 (e.g., PDL2 / Ig fusion proteins such as AMP 224), Gr1, or Ly6G), and iv) agents that directly block inhibitory pathways on cancer cells or rely on antibody-dependent cytotoxicity to enhance cytotoxic agents against cancer cells such as antibodies or small molecules (e.g., rituximab, antibodies or small molecules targeting PD-L1 (e.g., MPDL3280A / RG7446; MEDI4736; MSB0010718C; BMS 936559), and antibodies or small molecule inhibitors targeting B7-H3 (e.g., MGA271), B7-H4, Gal-9, or MUC1). In one embodiment, the inhibitor of the checkpoint is an inhibitor of HVEM, CD160, CHK 1, CHK2, B-7 family ligands, or a combination thereof (e.g., inhibitory antibody or small molecule inhibitor). Such agents as described herein can be designed and produced, for example, by conventional methods known in the art (e.g., Templeton, Gene and Cell Therapy, 2015; Green and Sambrook, Molecular Cloning, 2012). In one embodiment, the inhibitor of the checkpoint is an inhibitory antibody (e.g., a monospecific antibody such as a monoclonal antibody). The antibody can be, for example, humanized or fully human. In other embodiments, the inhibitor of the checkpoint is a fusion protein, e.g., an Fc-receptor fusion protein. In some embodiments, the inhibitor of the checkpoint is an agent that interacts with a checkpoint protein, such as an antibody. In other embodiments, the inhibitor of the checkpoint is an agent that interacts with a ligand of a checkpoint protein, such as an antibody.

[0400] Chemotherapeutic agents include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodophyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthraquinone-substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, adrenocortical steroids, progestins, estrogens, antiestrogens, androgens, antiandrogens, and gonadotropin-releasing hormone analogs. Also included are 5-fluorouracil (5-FU), leucovorin (LV), irinotecan, oxaliplatin, capecitabine, paclitaxel, and docetaxel. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog topotecan); bryostatin; spongistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenyl mustard chloromethyl cholesteryl, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γII and calicheamicin ωII; dynemicin, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoproteins enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, carubicin, carminomycin, carcinomycin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxy-doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin (such as mitomycin C), mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, triferrioxamycin, rhodomycin, streptothricin, streptozocin, tubercidin, bestatin, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as leucovorin, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioguanine, tioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, delmadinone acetate, epitiostanol, methylandrostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; glucuronolactone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; aclarubicin; bexarotene; bisantrene; edatrexate; defofamine; colchicine; diaziquone; eflornithine; elisidepsin; epsilon; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; melphalan; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazine; procarbazine; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichloroethylamine; trichothecenes (especially T-2 toxin, verrucarin A, roridin A and satratoxin); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulcitol; pipobroman; galcitabine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; nolatrexed; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine; ibandronate; iridium (e.g. CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine;and a pharmaceutically acceptable salt, acid or derivative of any of the foregoing. Two or more chemotherapeutic agents can be used in a mixture for administration in combination with the masked cells described herein. Suitable dosing regimens for combination chemotherapy are known in the art.;

[0401] Anticancer biologics include cytokines for cancer treatment (e.g., interferons or interleukins (e.g., IL-2)). In other embodiments, the biologic is an anti-angiogenic agent, such as an anti-VEGF agent, e.g., bevacizumab. In some embodiments, the biologic is an immunoglobulin-based biologic, e.g., a humanized antibody, a fully human antibody, an Fc fusion protein, or a functional fragment thereof), which agonizes a target to stimulate an anticancer response or antagonizes an antigen important for cancer. Such agents include rituximab; daclizumab; basiliximab; palivizumab; infliximab; trastuzumab; gemtuzumab ozogamicin; alemtuzumab; ibritumomab; adalimumab; omalizumab; tositumomab-I-131; efalizumab; cetuximab; bevacizumab; natalizumab; tocilizumab; panitumumab; ranibizumab; eculizumab; certolizumab pegol; golimumab; canakinumab; ustekinumab; ofatumumab; denosumab; motavizumab; raxibacumab; belimumab; ipilimumab; bintrafusp alfa; pertuzumab; ado-trastuzumab emtansine; and obinutuzumab. Antibody-drug conjugates are also included.

[0402] Kit

[0403] The invention also features a kit that contains the masked cells described herein (e.g., expressing a set of masked transgenes described herein (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 of PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8, and Spi6), optionally further expressing one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39 masked cells). In some embodiments, the masked cells are further modified to include one or more systems for regulating cell division (e.g., the ALINK or EARC system) and / or therapeutic agents (e.g., transgenes encoding proteins or antibodies). The masked cells can be provided in the form of a pharmaceutical composition. The kit can also include a syringe for administering the masked cells or the pharmaceutical composition and instructions for administering the masked cells or the pharmaceutical composition to treat the diseases or disorders described herein.

[0404] Examples

[0405] The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the present invention; it will be understood by their exemplary nature that other procedures, methods or techniques known to those skilled in the art may alternatively be used.

[0406] Example 1: Materials and Methods

[0407] Construction of vectors expressing target genes essential for allogeneic tolerance

[0408] Plasmids containing the cDNA sequences of genes involved in allogeneic tolerance were obtained as follows:

[0409] PD-L1: Mount Sinai Hospital, clone #V102001

[0410] FasL: Mount Sinai Hospital, #75719

[0411] Cd47: Mount Sinai Hospital, #V75535

[0412] Cd200: GEDharmacon, ID#17470

[0413] H2-M3: Mount Sinai Hospital, clone #8188

[0414] Ccl21: Mount Sinai Hospital, clone #V77120

[0415] Mfge8: Mount Sinai Hospital, clone #V72614

[0416] Spi6: Mount Sinai Hospital, clone #V8907

[0417] Expression vectors containing these genes of interest (GOI) or luciferase were generated using the Gateway cloning system (Thermo Fisher). Cd47, Ccl21, Mfge8, and Spi6 cDNAs were obtained in a form containing cDNA flanked by attB sites. For H2-M3, Cd200, FasL, and PD-L1, primers were designed to amplify the cDNA sequences and attB sites were added ( Figure 15 (a)). After PCR amplification, the attB-containing cDNA was recombined into the pDONR221 vector (Thermo Fisher, #1256017) by a BP (recombination between attB and attP sites) reaction to generate entry (pENTRY) clones ( Figure 15(b)). The BP reaction requires mixing the transgenic cDNA flanked by attB with the pDONR221 plasmid along with the buffer and BP enzyme provided by Invitrogen in a 1 mL tube, where the BP enzyme recombines the GOI into the docking site of the pDONR221 plasmid. The insertion of the transgene into the pDONR221 plasmid was verified by DNA sequencing (using the TCAG sequencing facility at the Centre for Applied Genomics, Toronto). Then, the pENTRY clone containing the GOI was recombined into the destination vector via the gateway LR (recombination between attL and attR sites) reaction ( Figure 15 (c)). The LR reaction requires mixing the pDONR221 plasmid containing the GOI and the destination vector along with the buffer and LR enzyme provided by Invitrogen in a 1 mL tube, where the LR enzyme recombines the GOI cassette from the pDONR221 plasmid into the docking site of the destination plasmid. The destination vector used for all transgenic constructs contains the CAGG promoter, followed by the Gateway entry site, an internal ribosome entry site (IRES), and a puromycin resistance selectable marker or a green fluorescent protein (GFP) reporter. The entire cassette is flanked by the transposable PB sites. After LR recombination, the final destination vector containing the GOI was verified by restriction enzyme digestion Figure 15 (d)).

[0418] ES cell culture, transfection, selection, and cloning

[0419] 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 cultures) 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). The cells were cultured on a feeder layer of mitomycin C-inactivated mouse embryonic fibroblasts (MEF). The cultures were maintained in a standard cell culture incubator at 37 °C and 5% CO2.

[0420] Transfection was performed using JetPRIME reagent (Polyplus, catalog number 14-07) according to the manufacturer's protocol and was carried out in three steps: 1) transfection with only the PD-L1-IRES-GFP destination vector, 2) transfection with all other transgenes carrying the puromycin selectable marker, and 3) transfection with the eLuciferase-IRES-GFP transgene.

[0421] Step 1: After transfection with PD-L1-IRES-GFP, cells were plated at low density such that after 5 - 6 days of multiple rounds of proliferation, single cell clones were selected based on the intensity of GFP expression - present as cell aggregates (colonies), and then expanded into clonal cell cultures. Clones with the highest and most consistent GFP expression were selected for the next step.

[0422] Step 2: 24 hours after transfection with a transgene containing the puromycin selection marker, puromycin was added to the medium. On the third day, cells were plated at clonal density and puromycin selection was continued until single colonies were picked and expanded into clones. A large number of these clones were screened in vivo, and clones that were able to form teratomas in an allogeneic environment were designated as "NT2".

[0423] Step 3: NT2 was transfected with PB-CAG-eLuciferase-IRES-GFP as described above and plated at clonal density. GFP+ clones were picked and expanded. 10 clones with high levels of GFP expression were selected for further study.

[0424] Evaluation of transgene expression levels

[0425] 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. The cell pellet was immediately frozen on dry ice and stored at -80 °C. Tumor tissues were 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 the standard protocol. cDNA was obtained by reverse transcriptase reaction using the Qiagen Quantifast Reverse Transcriptase Kit #205313. Quantitative PCR was performed using the Sensifast Master Mix from Bioline, #Bio-98020, gene-specific primers and 1:50 diluted RNA. Samples were plated in 384-well plates using the Eppendorf epMotion 5070 robot and quantitative PCR was performed on a BioRad CFX384 Real-Time System C1000 Thermal Cycler according to the standard protocol. qPCR data was captured by the BioRad CFXManager 3.1 software and expression levels were calculated using Microsoft Excel.

[0426] Teratoma assay

[0427] Matrigel Matrix High Concentration (Corning catalog number 354248) was diluted 1:1 with cold DMEM medium on ice. 5x107 The cells were suspended in 500uL of DMEM and an equal volume of Matrigel. 100ul of the suspension of 5x10 cells was subcutaneously injected into each dorsal flank of B6N (syngeneic) or FVB / N (allogeneic) mice. The resulting teratomas were formed 2-4 weeks after injection. The teratoma size was measured using a caliper and the volume was calculated using the formula V = (LxWxH) / 2. The tumor was allowed to grow to approximately 500mm 2 , which is a size that is well tolerated and also very suitable for downstream experiments. All transgenes are delivered into cells containing a "fail-safe system" (e.g., as described in WO 2016 / 141480, the entire contents of which are incorporated herein by reference. This genetic system allows complete inhibition of cell division by administration of ganciclovir (GCV). Once the teratomas from the aforementioned experiments reach 500 mm 2 , 50 mg / kg GCV was injected into the peritoneal cavity of mice every 2-3 days for 2-3 weeks. This treatment regimen resulted in an initial transient reduction in tumor size, which then stabilized at 400-500 mm after 2-3 weeks of treatment. 2 At the end of the experiment, mice were sacrificed and tumors were dissected. A small portion of the tissue was snap-frozen for RNA extraction, while the rest was fixed in 4% paraformaldehyde.

[0428] Bioluminescence imaging

[0429] Ten minutes before imaging, mice developing teratomas derived from cells transfected with the eLuciferase transgene were injected with 30 mg / mL of VivoGlo luciferin (Promega # P104C) at 100 uL / 25 g body weight. The animals were anesthetized with isoflurane and placed in an IVIS Lumina II imager (Caliper Life Sciences) driven by Living Image software. Depending on the signal intensity, the exposure time was set between 5 seconds and 5 minutes.

[0430] Histology

[0431] Fixed tumors were embedded in paraffin, sectioned and stained with hematoxylin / eosin for histological analysis at the CMHD pathology core. Histological images were processed with NDPview2 software.

[0432] Example 2: Generation of masked cells

[0433] Transgenes encoding the genes in Table 1 were cloned into expression vectors and sequence verified by polymerase chain reaction (PCR), restriction enzyme digestion, and sequencing, all using standard methods known in the art.

[0434] A set of constructs (Group 1) containing transgenic Cd47, Cd200, FasL, and H2-M3 was transfected into mouse embryonic stem cells derived from the inventors' C57BL / 6 mouse ES line (C2). The presence of the transgenes was verified by PCR, and the expression of the expressed proteins was documented by immunohistochemistry ( Figure 1A -D). A second set of constructs (Group 2) containing transgenic Ccl21, Mfge8, TGF-β, and Spi6 was transfected into ES cells (ES line C2) derived from FVB / N.

[0435] Except that the medium used was DMEM medium containing 10% fetal bovine serum (FBS), a similar method was used to generate masked B16F10 melanoma cells.

[0436] Example 3: Screening method for inhibiting T cell activation

[0437] The modified in vitro mixed lymphocyte reaction (MLR) assay was used to screen transgenic combinations to produce the most effective inhibition of T cell activation. Cell lines transfected with masked transgenes from Group 1 and Group 2 of Example 1 were used. Donor OT-1 splenocytes were labeled with carboxyfluorescein succinimidyl ester CFSE, and 60,000 cells were added to each well of a 96-well plate. ES or melanoma cells were mixed with egg cell-expressing cells at a ratio of 10:1. 10,000 of these were added to each well of the splenocytes. IL-2 was added as a general activator, and T cell proliferation was measured by flow cytometry after 3 days ( Figures 2A - 2E ). Initially, the cells were gated to include only CD8+ cells, and all conditions were set up in quadruplicate.

[0438] The negative control (only splenocytes) produced a baseline proliferation rate of 6.12% ( Figure 2A ). Wild-type B16 melanoma (+10% egg cell-expressing) cells led to a significant acceleration of proliferation to 17.1% ( Figure 2B ), while the masked cells reduced this proliferation to 9.51% ( Figure 2C ). Similar results were obtained for wild-type ( Figure 2E ) compared to masked ES cells ( Figure 2D ).

[0439] Example 4: Study on the use of WT and masked cancer cells in syngeneic and allogeneic transplanted B16F10 melanoma cells

[0440] Since some of the candidate masked transgenes are intended to inhibit or regulate the initial stages of the immune recognition cascade, the effects of these transgenes can be evaluated solely by MLR because these events act on the maturation and physical migration of host APCs to local lymph nodes, where they subsequently activate naive T and B cells.

[0441] This requires alternative assays that can screen a large number of transgenic combinations in an in vivo allogeneic environment. Intraperitoneal and intravenous injection of ES cells carrying multiple transgenic combinations was attempted as a selection. However, teratoma formation depends on the aggregation of a minimum number of ES cells (1x10 5 –5x10 6 , depending on the injection site), making this selection incompatible with such screening. However, the murine melanoma cell line B16F10 derived from C57BL / 6 mice is not limited in this way. Intravenous injection of less than 5x10 3 results in the effective induction of a large number of small cancer nodules in the lungs. By restricting the number of cells injected, it is expected that cancer cells trapped in the alveoli will form nodules derived from a single or very small number of cells. By isolating these nodules and genotyping them, the transgenes can be identified.

[0442] Injecting B16F10 melanoma cells into the bloodstream of C57BL / 6 mice (syngeneic graft control) results in the formation of cancer nodules in the lungs ( Figure 3A , left panel). However, when observed on day 14 after injection, small melanoma nodules also formed in the lungs of wild-type B16F10 melanoma transplanted into allogeneic control FVB mice as a negative control. However, when the melanoma was allowed to grow for 24 days, the nodules almost completely regressed ( Figure 3A , right panel).

[0443] The above experiment was repeated by injecting a mixture of cancer cells expressing random combinations of candidate shielding genes generated using the PiggyBac transposon system. Lung nodules developed in the allogeneic environment contained one or more successful combinations required to protect the allograft from recognition and rejection ( Figure 3B , right panel). The same immunologically shielded cells also caused accelerated development in syngeneic hosts ( Figure 3B , left panel).

[0444] Example 5: Unshielded embryonic stem cells do not form teratomas in an allogeneic environment

[0445] As shown in Table 4, it was confirmed that wild-type ESCs derived from C57BL / 6 mice cannot form teratomas in FVB / N mice. Similarly, it was also shown that wild-type ESCs derived from FVB / N background cannot form teratomas in C57BL / 6 hosts. ES cell colonies were dissociated with trypsin, washed once with DMEM without additives, and resuspended in Matrigel HC at a concentration of approximately 50 million cells per milliliter. Recipient mice were anesthetized and 100 microliters were subcutaneously injected into each flank area. Developing teratomas were followed for 12 weeks and verified by palpation and measuring the volume with calipers.

[0446] Table 4: Teratomas formed in FVB / N mice and C57BL / 6 hosts (injected with wild-type ESCs from C57BL / 6 mice or wild-type ESCs from FVB / N background)

[0447]

[0448] Example 6: Shielded ES cells can proliferate in syngeneic and allogeneic hosts

[0449] To verify the shielding ability of the candidate transgene, ESCs were transfected with the same transgene, and a luciferase transgene that can be detected by imaging was also added. Briefly, ES cells were prepared as described above. The presence of live cells was repeatedly measured by imaging. Figure 4 The images in were taken on day 17 after injection.

[0450] In Figure 4 the upper panel shows the proliferation of immunologically shielded cells in a syngeneic host, while the lower panel shows the proliferation of immunologically shielded cells in an allogeneic host.

[0451] In another experiment, shielded ES cells from C57BL / 6 mice with high expression of 8 immunomodulatory transgenes (clone NT2) were subcutaneously injected into different allogeneic mouse strains (C3H, FVB / N, and CD1) with mismatched MHC alleles. The red arrows indicate the formed teratomas ( Figures 5A - 5C ).

[0452] Example 7: Mice with shielded tissues are not immunocompromised

[0453] Non-immunologically shielded (wild-type) ESCs were transplanted into mice carrying pre-existing immunologically shielded tissues, and the mice were evaluated to determine whether they could effectively reject the non-immunologically shielded grafts ( Figure 6 ). The same mice were imaged several times over a 15-day period. As shown in the left panel of Figure 6 , in the syngeneic mouse control, the grafts were not rejected over time. For allogeneic FVB mice, the left mouse in the right panel of Figure 6 had a pre-existing immunologically shielded graft (arrow). In the middle mouse in the right panel of Figure 6 , C57BL / 6 allogeneic ESCs had been previously implanted, but the grafts were rejected (although not limited to theory, the rejection may be due to pre-formed antibodies against C57BL / 6 cells). Figure 6The mouse on the right in the right figure has never been transplanted before. All three mice successfully rejected the non-immunoprotected grafts. The mouse on the right rejected the graft more slowly, probably because it did not have any preformed antibodies against C57BL / 6 cells.

[0454] Similar experiments were conducted in which wild-type embryonic stem cells were detected up to 9 days after injection into FVB / N mice with protected teratomas ( Figure 7 ). However, on day 12, no evidence of remaining cells was detected. Control animals were C57BL / 6 mice that also carried protected tumors. The signal in these mice increased over the course of the experiment.

[0455] Example 8: Protected and fail-safe embryonic stem cell lines

[0456] When the fail-safe C57BL / 6 ES cell line (e.g., as described in WO / 2016 / 141480) was co-transfected with 5 candidate protective transgenes (PD-L1, FasL, Cd47, Cd200, and H2-M3), none of these transgenic lines produced teratomas in an allograft environment. When the group of co-transfected genes was amplified by three additional candidate protective genes: Spi6, Ccl21b, and Mfge8, 38 clonal lines were generated. One of these lines, NT2, produced a teratoma in an allogeneic recipient (FVB). The expression levels of the protective genes in 38 clonal lines (including the NT2 line) were measured using quantitative PCR (see Figures 8A - 8H the arrow in Figures 8A - 8H ). Among the 38 clones, NT2 was 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-expressing factor among the clones, its expression was also significantly increased relative to the 350x expression of ES cells. The expression of these genes was also examined in the Project Grandiose dataset (www.stemformatics.org / project_grandiose), and it was found that the expression of Ccl21b, FasL, Cd200, PD-L1, and Spi6 was below the detection threshold, so their expression relative to ES cells was very high. Based on this data, these eight highly activated genes may have a major role in inducing immune tolerance of allografts.

[0457] NT2 cells were injected into C57BL / 6 in an FVB allogeneic environment ( Figures 11A - 11B) and teratomas are generated in the FVB isoC57Bl / 6 allogeneic environment. From day 12 to day 38, allogeneic teratomas (n = 6) grew steadily. At a size of 500 mm ...

Claims

1. A cell that has been genetically modified to contain a transgene, the transgene comprising: (a) major histocompatibility complex class I G (HLA-G), CD47, CD200, and human Fas ligand (FASLG); (b) C-C motif chemokine ligand 21 (CCL21), milk fat globule-EFG factor 8 (MFGE8), transforming growth factor β (TGF-β), and serine protease inhibitor family B member 9 (SERPINB9), (c) histocompatibility 2M region locus 3 (H2-M3), Cd47, Cd200, and murine Fas ligand (FasL); or (d) Ccl21b, Mfge8, TGF-β, and serine protease inhibitor 6 (Spi6).

2. The cell of claim 1, wherein: the human FASLG transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 10, the human CD200 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 6, the human CCL21 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 2, the human SERPINB9 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 8, the human MFGE8 transgene encodes a protein having at least 90% identity with the sequence set forth in SEQ ID NO: 14, the human PD-L1 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 12, the human HLA-G transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 16, and the human CD47 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 4; or the murine FasL transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 9, the murine Cd200 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 5, the murine Ccl21b transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 1, the murine Spi6 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 7, the murine Mfge8 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 13, the murine Pd-l1 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 11, the murine H2-M3 transgene encodes a protein having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 15, or A murine Cd47 transgene encodes a protein having at least 90% sequence identity to the sequence set forth in SEQ ID NO:

3.

3. The cell according to claim 1, wherein the cell further comprises a heterologous sequence encoding a therapeutic agent.

4. The cell according to claim 3, wherein the therapeutic agent comprises the wild-type form of a gene mutated in a subject.

5. The cell according to claim 3, wherein the therapeutic agent comprises an enzyme, an antibody, a growth factor, or a cytokine.

6. The cell according to claim 1, wherein the expression of the transgene is under the control of one or more constitutive promoters.

7. The cell according to claim 1, wherein the cell further comprises one or more of the following transgenes: ecto-5'-nucleotidase (Cd73), ectonucleoside triphosphate diphosphohydrolase 1 (Cd39), lymphocyte activation 3 (Lag3), interleukin 1 receptor type 2 (Il1r2), atypical chemokine receptor 2 (Ackr2), tumor necrosis factor receptor superfamily member 22 (Tnfrsf22), tumor necrosis factor receptor superfamily member 23 (Tnfrsf23), tumor necrosis factor receptor superfamily member 10 (Tnfrsf10), anti-cell death defensin 1 (Dad1), or a dominant negative form of the IFNγ receptor (IFNγR1 d39).

8. The cell according to claim 1, wherein the cell further comprises a genetic modification of one or more cell division locus / loci (CDL).

9. A composition comprising the cell according to claim 1 and a pharmaceutically acceptable excipient.

10. A method of inhibiting T cell activation, the method comprising: exposing T cells to a cell genetically modified to express a transgene selected from: (i) major histocompatibility complex class I G (HLA-G) or histocompatibility 2 M region locus 3 (H2-M3), Cd47, Cd200, and human Fas ligand (FASLG) or murine Fas ligand (FasL); or (ii) C-C motif chemokine ligand 21 (CCL21) or Ccl21b, milk fat globule-EFG factor 8 (MFGE8), transforming growth factor β (TGF-β), and serine protease inhibitor family B member 9 (SERPINB9) or serine protease inhibitor 6 (Spi6), thereby inhibiting T cell activation.

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