US20210161971A1 - Allograft tolerance without the need for systemic immune suppression - Google Patents

Allograft tolerance without the need for systemic immune suppression Download PDF

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US20210161971A1
US20210161971A1 US16/621,490 US201816621490A US2021161971A1 US 20210161971 A1 US20210161971 A1 US 20210161971A1 US 201816621490 A US201816621490 A US 201816621490A US 2021161971 A1 US2021161971 A1 US 2021161971A1
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Andras Nagy
Jeffrey Harding
Kristina NAGY
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Sinai Health System
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Definitions

  • the disclosure relates generally to the field of transplantation.
  • the disclosure further relates to methods for generating local immunosuppression in the environment of transplanted cells.
  • ES embryonic stem
  • iPS induced pluripotent stem
  • Immune rejection remains a critical barrier 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 mouse, HLA in humans)—that differ between donor and recipient (Yang et al., Nat Rev Genet. 18:309-26 (2017)).
  • MHC major histocompatibility complex
  • HLA major histocompatibility complex
  • rejection of transplanted cells or tissues can occur over the timescale of minutes/hours (hyperacute), days/months (acute), and months/years (chronic) (LaRosa et al., J Immunol. 178:7503-9 (2007)).
  • This rejection results from the complex and coordinated effects of cell types from both innate (Murphy et al., Immunol Rev. 241:39-48 (2011)) and adaptive immunity (Issa et al., Expert Rev Clin Immunol. 6:155-69 (2010)).
  • T-cells One of the most important pathways to rejection is the priming of the adaptive immune system and 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 lymph organs (Lechler et al., J Exp Med. 155:31-41 (1982); Guermonprez et al., Annu Rev Immunol. 20:621-67 (2002); Stockwin et al., Immunol Cell Biol. 78:91-102 (2000)). These T-cells then migrate to and kill transplanted cells or tissues with the release of cytolytic factors like perforin and granzyme.
  • NK-cells can also induce apoptosis in donor cells based on foreign or no MHC expression (Kitchens et al., Transplantation. 81:811-7 (2006); Benichou et al., Curr Opin Organ Transplant. 16:47-53 (2011)), and other cell types like macrophages can support rejection with the release of pro-inflammatory cytokines at the engraftment site (Mannon, Curr Opin Organ Transplant. 17:20-5 (2012)). Many other cell types and subtypes also have a role in allograft rejection. Since these are the same immune pathways used to eliminate common viral and bacterial pathogens, they are—along with rejection of an allograft—highly conserved across vertebrate species.
  • a current solution to prevent rejection of an allograft involves the following two options: find a donor with a matched histocompatibility haplotype (mostly likely from genetically-related family), and much more commonly, use broadly-directed immunosuppressant drugs (Wiseman, Clin J Am Soc Nephrol. 11:332-43 (2016); Malaise et al., Transplant Proc. 37:2840-2 (2005)).
  • Common drugs include those from the families of calcineurin inhibitors (Flechner et al., Clin Transplant. 22:1-15 (2008); Casey et al., Curr Opin Nephrol Hypertens. 20:610-5 (2011)), anti-proliferative agents (Hardinger et al., World J Transplant.
  • iPS cells could be created from, and for, each patient. These cells should, in theory, be protected from immune rejection by the corresponding patient (Pearl et al., Sci Transl Med. 4:164p525 (2012)).
  • the induction of an iPS cell state involves epigenetic alterations and in-vitro-culture pressures that can create abnormalities and malignancies, so each cell line would need to be vigorously tested and/or genetically modified to achieve safety as well as function (Hussein et al., Nature. 471:58-62 (2011); Laurent et al., Cell Stem Cell. 8:106-18 (2011); Lister et al., Nature. 471:68-73 (2011)).
  • Tregs may promote tolerance by a number of mechanisms, including (but not limited to) the expression of suppressive factors like TGF beta (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 ( mmunol. 36:63-70 (2015)), IL10 (O'Garra et al., J Clin Invest. 114:1372-8 (2004); Chaudhry et al., Immunity.
  • suppressive factors like TGF beta (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);
  • Tregs Two approaches for potential therapeutic uses of Tregs involve either in-vitro expansion using donor antigens coupled with transplantation, or selective in-vivo expansion that leverages differences between regulatory and effector T-cells. While these strategies are interesting, to date no long term of acceptance of an allograft has been demonstrated solely with the use of in-vitro or in-vivo expanded Tregs. There remain many complications and unknown facets to Treg biology, including the optimal methodology for in-vitro or in-vivo expansion, as well as the therapeutically-relevant dosage and timing. It has also been shown that antigen-specific Treg suppression can be “defeated” depending on the inflammatory context (Korn et al., Nat Med. 13:423-31 (2007)) and that Tregs can be killed by NK-cells (Roy et al., J Immunol. 180:1729-36 (2008)).
  • DCs dendritic cells
  • DCs present allograft antigens inside the binding grooves of MHC (mouse) or HLA (human) molecules on their surface, along with costimulatory molecules like CD80, CD86, and CD40 (among others), which allograft-specific T-cell clones recognize to become activated (Walker et al., Trends Immunol. 36:63-70 (2015)).
  • Tolerogenic DCs can be induced from the immature state by exposure to suppressive cues, which keep expression levels of MHC and costimulatory molecules low and in turn promote na ⁇ ve T-cells into anergic or even Tregulatory subtypes upon DC-Tcell interactions.
  • one application of this biology is to expand DCs in vitro exposed simultaneously to specific allograft antigens of interest and immunosuppressive factors—many of which have been tested including TGF-beta, IL10, cAMP, prostaglandin E2, histamine, neuropeptides, vitamin D2, B2 agonists, HLA-G, glucosamine, as well drugs like corticosteroids, cyclosporine, tacrolimus, rapamycin, aspirin, mecophenolate mofetil, sanglifehrin, and deoxyspergualin (Hackstein et al., Nat Rev Immunol. 4:24-34 (2004)).
  • DCs have been genetically engineered to directly express immunomodulatory factors like TGF-beta, IL-10, VEGF, FasL, CTLA4-Ig, IDO, NFKb decoy receptors, soluble TNFR, CCR7, as well as siRNA-induced silencing of IL-12 (Morelli et al., Immunol Rev. 196:125-46 (2003)).
  • immunomodulatory factors like TGF-beta, IL-10, VEGF, FasL, CTLA4-Ig, IDO, NFKb decoy receptors, soluble TNFR, CCR7, as well as siRNA-induced silencing of IL-12 (Morelli et al., Immunol Rev. 196:125-46 (2003)).
  • These cultured or engineered DCs are then transferred into recipients concomitantly with an allograft to test whether they can prolong the survival of an allograft, with the assumption that they suppress allograft-specific T-cells
  • bone-marrow derived DCs were transduced with SOCS1 (preventing upregulation of costimulatory molecules and MHCII), which prolonged mouse cardiac allografts (Fu et al., Cell Mol Immunol. 6:87-95 (2009)).
  • FasL-expressing DCs were also able to prolong mouse cardiac allografts (Min et al., J Immunol. 164:161-7 (2000)).
  • tolerogenic DCs along these lines.
  • HCT Hematopoietic Cell transplantation
  • HCT Since its inception, several variants of HCT to dampen rejection have been developed, including the use of nonmyeloablative strategies. These strategies use altered chemotherapy regimens, often involving lower dosages, so that the recipient receiving the HCT does not receive total ablation of their hematopoietic compartment. The most recent of these strategies, for instance, used a tolerance-promoting facilitating cell (FC)-based HCT to promote tolerance in HLA-mismatched kidney recipients while largely avoiding GHVD (Leventhal et al., Sci Transl Med. 4:124ra28 (2012)).
  • FC tolerance-promoting facilitating cell
  • CTLA4-Ig can lead to systemic immune suppression. It has not yet been demonstrated that a set of modifications to ES or iPS cells allows them to escape allorejection without the potential for systemic immunosuppression and without the need for immunosuppressive drugs.
  • a cell genetically modified to comprise at least one mechanism for providing a local immunosuppression at a transplant site when transplanted in an allogeneic host comprises: a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting, and has one or more of the following functions: a) to mitigate antigen presenting cell activation and function; b) to mitigate graft attacking leukocyte activity or cytolytic function; c) to mitigate macrophage cytolytic function and phagocytosis of allograft cells; d) to induce apoptosis in graft attacking leukocytes; e) to mitigate local inflammatory proteins; and f) to protect against leukocyte-mediated apoptosis.
  • the set 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 (or the mouse version of HLA-G, H2-M3), Cd47, Cd200, FASLG (or the mouse version of FASLG, FasL), Ccl21 (or the mouse version of Ccl21, Ccl21b), Mfge8, and Serpin B9 (or the mouse version of Serpin B9, Spi6).
  • PD-L1, HLA-G or the mouse version of HLA-G, H2-M3
  • Cd47, Cd200 FASLG (or the mouse version of FASLG, FasL)
  • Ccl21 or the mouse version of Ccl21, Ccl21b
  • Mfge8 or the mouse version of Serpin B9, Spi6
  • the set 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).
  • the set of transgenes genes comprises PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) or a gene encoding a biologic that acts as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).
  • 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- ⁇ , Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFN ⁇ R1 d39 or a gene encoding a biologic that acts as an agonist of TGF- ⁇ , Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFN ⁇ R1 d39.
  • the TGF- ⁇ or the biologic is local acting in the graft environment. In an embodiment of the cell, the TGF- ⁇ or the biologic is local acting in the graft environment with minimal systemic effect.
  • the cell is a stem cell, a cell amenable for genome editing, and/or a source of a therapeutic cell type (e.g., a cell that can be differentiated into a therapeutic cell type, or a cell of a desired target tissue).
  • a therapeutic cell type e.g., a cell that can be differentiated into a therapeutic cell type, or a cell of a desired target tissue.
  • the cell is an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem or progenitor cell, a germline stem cell, a lung stem or progenitor cells, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, an intestinal stem or progenitor cell, a multipotent stem cell, an amniotic stem cell, a cord blood stem cell, a neural stem or progenitor cell, an adult stem cell, a somatic stem cell, a tissue-specific stem cell, a totipotent stem cell, a fibroblast, a monocytic precursor, a B cell, an exocrine cell, a pancreatic progenitor, an endocrine progenitor, a he
  • the cell is further genetically modified to comprise at least one (e.g., one, two, three, or more) mechanism for controlling cell proliferation (e.g., to reduce the tumorigenic potential of the modified cell or to reduce proliferation of a modified cell that has become tumorigenic).
  • at least one e.g., one, two, three, or more
  • mechanism for controlling cell proliferation e.g., to reduce the tumorigenic potential of the modified cell or to reduce proliferation of a modified cell that has become tumorigenic.
  • the genetically modified cell comprises: a genetic modification of one or more (e.g., one, two, three, or more) cell division locus/loci (CDL), the CDL being one or more loci whose transcription product(s) is expressed by dividing cells (e.g., all dividing cells containing one or more of the immunosuppressive transgenes), the genetic modification being one or more of: a) an ablation link (ALINK) system, the ALINK system comprising a DNA sequence encoding a negative selectable marker that is transcriptionally linked to a DNA sequence encoding the CDL; and b) an exogenous activator of regulation of a CDL (EARC) system, the EARC system comprising an inducible activator-based gene expression system that is operably linked to the CDL.
  • CDL cell division locus/loci
  • the genetic modification of the CDL comprises performing targeted replacement of the CDL with one or more of: a) a DNA vector comprising the ALINK system; b) a DNA vector comprising the EARC system; and c) a DNA vector comprising the ALINK system and the EARC system; wherein the ALINK and/or EARC systems are each operably linked to the CDL.
  • 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 generated through EARC-modified alleles.
  • the CDL is one or more (e.g., one, two, three, or more) of the loci recited in Table 5.
  • the CDL encodes a gene product that functions in one or more of: cell cycle, DNA replication, RNA transcription, protein translation, and metabolism.
  • 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.
  • the CDL is Top2A.
  • the CDL is Eef2.
  • 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.
  • the ALINK system comprises a herpes simplex virus-thymidine kinase/ganciclovir system, a cytosine deaminase/5-fluorocytosine system, a carboxyl esterase/irinotecan system or an iCasp9/AP1903 system, preferably the ALINK system is a herpes simplex virus-thymidine kinase/ganciclovir system.
  • the EARC system is a doxycycline inducible “dox-bridge” system, a cumate 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.
  • a method for providing a local immunosuppression at a transplant site in an allogeneic host comprises providing a cell; and expressing in the cell a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting, and has one or more of the following functions: a) to mitigate antigen presenting cell activation and function; b) to mitigate graft attacking leukocyte activity or cytolytic function; c) to mitigate macrophage cytolytic function and phagocytosis of allograft cells; d) to induce apoptosis in graft attacking leukocytes; e) to mitigate local inflammatory proteins; and f) to protect against leukocyte-mediated apoptosis.
  • the set 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 (Ccl21 b), Mfge8, and Serpin B9 (Spi6) or a gene encoding a biologic that acts as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21 b), Mfge8, or Serpin B9 (Spi6).
  • genes e.g., one, two, three, four, five, six, seven, or all eight
  • the set 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).
  • the set of transgenes genes comprises PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21 b), Mfge8, and Serpin B9 (Spi6) or a gene encoding a biologic that acts as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21 b), Mfge8, and Serpin B9 (Spi6).
  • 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- ⁇ , Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFN ⁇ R1 d39 or a gene encoding a biologic that acts as an agonist of TGF- ⁇ , Cd73, Cd39, Lag3, 111r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFN ⁇ R1 d39.
  • the TGF- ⁇ or the biologic is local acting in the graft environment.
  • the TGF- ⁇ or the biologic is local acting in the graft environment with minimal systemic effect.
  • the cell is a stem cell, a cell amenable to genome editing, and/or a source of a therapeutic cell type (e.g., a cell that can be differentiated into a therapeutic cell type, or a cell of a desired target tissue).
  • a therapeutic cell type e.g., a cell that can be differentiated into a therapeutic cell type, or a cell of a desired target tissue.
  • the cell is an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem or progenitor cell, a germline stem cell, a lung stem or progenitor cells, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, an intestinal stem or progenitor cell, a multipotent stem cell, an amniotic stem cell, a cord blood stem cell, a neural stem or progenitor cell, an adult stem cell, a somatic stem cell, a tissue-specific stem cell, a totipotent stem cell, a fibroblast, a monocytic precursor, a B cell, an exocrine cell, a pancreatic progenitor, an endocrine progenitor, a he
  • the cell is provided (e.g., injected) to or near the transplant site.
  • the cell is provided (e.g., injected or implanted) into the transplant (e.g., injected or implanted into the tissue or organ transplant before, during, or after transplantation).
  • the cell in which the transgenes are expressed is a cell of the transplant (e.g., a cell of the tissue or organ that is being transplanted is modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)).
  • PD-L1, HLA-G (H2-M3) e.g., one, two, three, four, five, six, seven, or all eight
  • HLA-G H2-M3
  • Cd47 Cd200
  • FASLG FasL
  • Ccl21 Ccl21b
  • Mfge8 Serpin B9
  • a method of controlling proliferation of a cell at a transplant site in an allogeneic host is provided (e.g., to reduce the tumorigenic potential of the cell at the transplant site or to reduce proliferation of the cell that has become tumorigenic at the transplant site).
  • the method comprises: a) genetically modifying in the cell one or more (e.g., one, two, three, or more) cell division locus/loci (CDL), the CDL being one or more loci whose transcription product(s) is expressed by dividing cells (e.g., all dividing cells containing one or more of the immunosuppressive transgenes), the genetic modification of the CDL comprising one or more of: i) an ablation link (ALINK) system, the ALINK system comprising a DNA sequence encoding a negative selectable marker that is transcriptionally linked to a DNA sequence encoding the CDL; and i) an inducible exogenous activator of regulation of a CDL (EARC) system, the EARC system comprising an inducible activator-based gene expression system that is operably linked to the CDL; b) genetically modifying the cell to comprise at least one mechanism for providing a local immunosuppression at a transplant site; c) transplanting the cell or a population of the cells at a
  • the genetic modification of the CDL comprises performing targeted replacement of the CDL with one or more of: a) a DNA vector comprising the ALINK system; b) a DNA vector comprising the EARC system; and c) a DNA vector comprising the ALINK system and the EARC system; wherein the ALINK and/or EARC systems are each operably linked to the CDL.
  • 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 generated through EARC-modified alleles.
  • the CDL is one or more (e.g., one, two, three, or more) of the loci recited in Table 5.
  • the CDL encodes a gene product whose function is involved with one or more of: cell cycle, DNA replication, RNA transcription, protein translation, and metabolism.
  • 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.
  • the CDL is Top2A.
  • the CDL is Eef2.
  • 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.
  • the ALINK system comprises a herpes simplex virus-thymidine kinase/ganciclovir system, a cytosine deaminase/5-fluorocytosine system, a carboxyl esterase/irinotecan system or an iCasp9/AP1903 system, preferably the ALINK system is a herpes simplex virus-thymidine kinase/ganciclovir system.
  • the EARC system is a doxycycline inducible “dox-bridge” system, a cumate 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.
  • the genetically modified cell comprises: a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting and has one or more of the following functions: a) to mitigate antigen presenting cell activation and function; b) to mitigate graft attacking leukocyte activity or cytolytic function; c) to mitigate macrophage cytolytic function and phagocytosis of allograft cells; d) to induce apoptosis in graft attacking leukocytes; e) to mitigate local inflammatory proteins; and f) to protect against leukocyte-mediated apoptosis.
  • the set 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 biologic that acts as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, or Serpin B9 (Spi6).
  • PD-L1, HLA-G (H2-M3) Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, or Serpin B9 (Spi6).
  • the set 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).
  • the set of transgenes genes comprises PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) or a gene encoding a biologic that acts a as an agonist of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).
  • 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- ⁇ , Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFN ⁇ R1 d39 or a gene encoding a biologic that acts as an agonist of TGF- ⁇ , Cd73, Cd39, Lag3,111r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFN ⁇ R1 d39.
  • TGF- ⁇ e.g., one, two, three, four, five, six, seven, eight, nine, ten, or all eleven
  • transgenes e.g., one, two, three, four, five, six, seven, eight, nine, ten, or all eleven
  • the TGF- ⁇ or the biologic is local acting in the graft environment. In an embodiment, the TGF- ⁇ or the biologic is local acting in the graft environment with minimal systemic effect
  • the cell is a stem cell, a cell amenable to genome editing, and/or a source of therapeutic cell type (e.g., a cell that can be differentiated into a therapeutic cell type, or a cell of a desired target tissue).
  • a source of therapeutic cell type e.g., a cell that can be differentiated into a therapeutic cell type, or a cell of a desired target tissue.
  • the cell is an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem or progenitor cell, a germline stem cell, a lung stem or progenitor cells, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, an intestinal stem or progenitor cell, a multipotent stem cell, an amniotic stem cell, a cord blood stem cell, a neural stem or progenitor cell, an adult stem cell, a somatic stem cell, a tissue-specific stem cell, a totipotent stem cell, a fibroblast, a monocytic precursor, a B cell, an exocrine cell, a pancreatic progenitor, an endocrine progenitor, a he
  • the cell is provided (e.g., injected) to or near the transplant site.
  • the cell is provided (e.g., injected or implanted) into the transplant (e.g., injected or implanted into the tissue or organ transplant before, during, or after transplantation).
  • the cell in which the transgenes are expressed is a cell of the transplant (e.g., a cell of the tissue or organ that is being transplanted is modified to express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)).
  • PD-L1, HLA-G (H2-M3) e.g., one, two, three, four, five, six, seven, or all eight
  • HLA-G H2-M3
  • Cd47 Cd200
  • FASLG FasL
  • Ccl21 Ccl21b
  • Mfge8 Serpin B9
  • the allogeneic host is a mammal. In various embodiments of the method, the allogeneic host is a mouse or a human.
  • the host has a degenerative disease or condition that can be treated with cell therapy.
  • the disease or condition is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., Type 1 or Type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, a neurological disease, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS, an enzyme or hormone deficiency, a metabolic disorder (e.g., a lysosomal storage disorder, Galactosemia, Maple syrup urine disease, Phenylketonuria, a glycogen storage disease, a mitochondrial disorder, Friedrich's ataxia, a peroxisomal disorder, a metal metabolism disorder, or an organic academia), an autoimmune disease (e.g., Psoriasis, Systemic Lupus Erythematosus, Grave's disease, Inflamm
  • arthritis e.g., osteo
  • 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) is expressed at a level that is equal to or greater than the expression level of the corresponding endogenous gene in an activated leukocyte (e.g., a T cell, e.g., the expression level of the cloaking transgene is equal to the level of expression of the endogenous gene in activated leukocytes, or is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more higher than the level of expression of the endogenous gene in activated leukocytes).
  • an activated leukocyte e.g., a T cell, e.g., the expression level of the cloaking transgene is equal to the level of expression of the endogenous gene in activate
  • all eight of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level that is equal to or greater than the expression level of the corresponding endogenous gene in an activated leukocyte.
  • 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) is expressed at a level that is greater than the expression level of the corresponding endogenous gene in a wild-type stem cell (e.g., a wild-type ES cell from the same species, e.g., the expression level of the cloaking transgene is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1,000-fold or more higher in cloaked cells compared to expression of the endogenous gene in unmodified wild-type ES cells from the same species).
  • a wild-type stem cell e.g., a wild-type ES cell from the same species, e.g., the expression level of the cloaking transgen
  • 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 that is greater (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100-fold higher or more) than the expression level of the endogenous gene in a wild-type stem cell (e.g., an embryonic stem cell from the same species as the cloaked cell).
  • a wild-type stem cell e.g., an embryonic stem cell from the same species as the cloaked cell.
  • 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) is expressed at a level that is in the top 5% of gene expression for all genes in the ES cell genome.
  • 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) is expressed at a level that is in the top 1% of gene expression for all genes in the ES cell genome.
  • all of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) are expressed at a level that is in the top 5% of gene expression for all genes in the ES cell genome.
  • the PD-L1 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.
  • the HLA-G (H2-M3) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 15.
  • the Cd47 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
  • the CD200 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6.
  • the FASLG (FasL) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 9.
  • the Ccl21 (Ccl21b) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1.
  • the Mfge8 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
  • the Serpin B9 (Spi6) transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 7.
  • the IFN ⁇ R1 d39 transgene encodes a protein having at least 85% identity (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or more) to the amino acid sequence of SEQ ID NO: 17.
  • the one or more transgenes is operably linked to a constitutive promoter.
  • the constitutive promoter is selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1 ⁇ promoter, the 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 the Rous sarcoma virus (RSV) promoter.
  • CMV cytomegalovirus
  • PGK adenovirus late promoter
  • vaccinia virus 7.5K promoter vaccinia virus 7.5K promoter
  • SV40 promoter vaccinia virus 7.5K promoter
  • SV40 promoter vaccinia virus 7.5K promoter
  • SV40 promoter vaccinia virus
  • the cell further comprises (e.g., the cell is further modified to include) a transgene encoding a therapeutic agent.
  • the therapeutic agent is a protein or antibody.
  • the antibody is an inhibitory antibody or agonist antibody.
  • the therapeutic agent is an agent listed in Table 2.
  • the therapeutic agent is the wild-type version of a gene that is mutated in the subject (e.g., the wild-type version of the mutated gene that is associated with the disease or condition in the subject, e.g., a genetic mutation that is associated with cancer, an enzyme or hormone deficiency, a metabolic disorder, or a degenerative disease).
  • the therapeutic agent is expressed using an inducible expression system selected from the group consisting of a tetracycline response element, a light inducible system, a radiogenetic system, a cumate switch inducible system, an ecdysone inducible system, a destabilization domain system, or a ligand-reversible dimerization system.
  • an inducible expression system selected from the group consisting of a tetracycline response element, a light inducible system, a radiogenetic system, a cumate switch inducible system, an ecdysone inducible system, a destabilization domain system, or a ligand-reversible dimerization system.
  • the therapeutic agent is expressed using a constitutive promoter selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1 ⁇ promoter, the 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 the Rous sarcoma virus (RSV) promoter.
  • a constitutive promoter selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1 ⁇ promoter, the 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
  • a method for providing a local immunosuppression at a transplant site in an allogeneic host comprises transplanting a genetically modified cell as described above or a population of genetically modified cells as described above at a transplantation site in an allogeneic host.
  • the invention features a composition containing a cell of the invention.
  • the composition further includes a pharmaceutically acceptable excipient.
  • kits including a cell of the invention or a pharmaceutical composition of the invention.
  • the disease or condition is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., Type 1 or Type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, a neurological disease, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS, an enzyme or hormone deficiency, a metabolic disorder (e.g., a lysosomal storage disorder, Galactosemia, Maple syrup urine disease, Phenylketonuria, a glycogen storage disease, a mitochondrial disorder, Friedrich's ataxia, a peroxisomal disorder, a metal metabolism disorder, or an organic academia), an autoimmune disease (e.g., Psoriasis, Systemic Lupus
  • the disease or condition is age-related macular degeneration (e.g., wet AMD) or retinal dystrophy and the therapeutic agent is a VEGF inhibitor (e.g., a soluble form of a VEGF receptors (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.
  • a VEGF inhibitor e.g., a soluble form of a VEGF receptors (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 Nat
  • the disease or condition is osteoarthritis or rheumatoid arthritis and the therapeutic agent is an anti-inflammatory biologic (e.g.
  • a TNF ⁇ 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
  • another agent used to treat rheumatoid arthritis e.g., abatacept, rituximab
  • the disease or condition is diabetes (e.g., Type 1 diabetes or Type 2 diabetes) and the therapeutic agent is insulin.
  • the disease or condition is hemophilia and the therapeutic agent is Factor VIII.
  • the disease or condition is a metabolic deficiency and the therapeutic agent is a transgene having the nucleic acid sequence of the wild-type version of the gene that is mutated in the subject or a transgene encoding an enzyme that is deficient in the subject.
  • the cells are differentiated into a lineage restricted cell type prior to administration to the subject.
  • the disease or condition is myocardial infarction and the cells are differentiated into cardiac muscle cells.
  • the disease or condition is blindness and the cells are differentiated into photoreceptor cells.
  • the disease or condition is spinal cord injury, Parkinson's disease, Huntington's disease, or Alzheimer's disease and the cells are dissociated into neurons.
  • the disease or condition is multiple sclerosis and the cells are differentiated into glial cells.
  • the cells are administered (e.g., injected or implanted) locally to the tissue or body site in need of cells or the therapeutic agent.
  • the cells are administered intravenously, subcutaneously, intramuscularly, percutaneously, intradermally, parenterally, intraarterially, intravascularly, or by perfusion.
  • the cells are administered by subcutaneous injection to produce a cloaked subcutaneous tissue.
  • the cells are administered as a tissue.
  • the tissue is administered with a gel, biocompatible matrix, or cellular scaffold.
  • the cells are administered in an amount of 25,000 to 5,000,000,000 cells (e.g., 2.5 ⁇ 10 4 , 5 ⁇ 10 4 , 7.5 ⁇ 10 4 , 1 ⁇ 10 5 , 2 ⁇ 10 5 , 3 ⁇ 10 5 , 4 ⁇ 10 5 , 6 ⁇ 10 5 , 6 ⁇ 10 5 , 7 ⁇ 10 5 , 8 ⁇ 10 5 , 9 ⁇ 10 5 , 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 7 ⁇ 10 8 , 7 ⁇ 10 8
  • the cells are administered in an amount of 800,000,000 to 100,000,000,000 cells (e.g., 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , or 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 1 ⁇ 10 10 , 2 ⁇ 10 10 , 3 ⁇ 10 10 , 4 ⁇ 10 10 , 5 ⁇ 10 10 , 6 ⁇ 10 10 , 7 ⁇ 10 10 , 8 ⁇ 10 10 , 9 ⁇ 10 10 , or 1 ⁇ 10 11 , cells).
  • 800,000,000 to 100,000,000,000 cells e.g., 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , or 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 9 , 7 ⁇ 10 10 , 8 ⁇ 10 10 , 9 ⁇ 10 10 , or 1 ⁇ 10 11 , cells).
  • the method further includes administering an additional therapeutic agent.
  • the additional therapeutic agent is administered prior to administration of the cells.
  • the additional therapeutic agent is administered after administration of the cells.
  • the additional therapeutic agent is administered concurrently with administration of the cells.
  • the additional therapeutic agent is an immunosuppressive agent, a disease-modifying anti-rheumatic drug (DMARD), a biologic response modifier (a type of DMARD), a corticosteroid, or a nonsteroidal anti-inflammatory medication (NSAID), prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, tofacitinib, adalimumab, abatacept, anakinra, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab or tocilizumab, 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab, an aminosalicylate, an antibiotic
  • the method further comprises controlling proliferation of the cell.
  • the cell comprises an ALINK system
  • the method of controlling proliferation comprises: i) permitting proliferation of the cell comprising the ALINK system by maintaining the cell comprising the ALINK system in the absence of an inducer of the negative selectable marker; or ii) ablating or inhibiting proliferation of the cell comprising the ALINK system by exposing the cell comprising the ALINK system to the inducer of the negative selectable marker.
  • the cell comprises an EARC system
  • the method of controlling cell proliferation comprises: i) permitting proliferation of the cell comprising the EARC system by exposing the cell comprising the EARC system to an inducer of the inducible activator-based gene expression system; or ii) preventing or inhibiting proliferation of the cell comprising the EARC system by maintaining the cell comprising the EARC system in the absence of the inducer of the inducible activator-based gene expression system.
  • the cell is removed after completion of the therapy. Removal of the cell(s) can be by surgery (e.g., to remove transplanted tissue or organs, or to remove cloaked subcutaneous tissue) or by the use of the ALINK and/or EARC systems. In some embodiments, one or more (e.g., one, two, three, four, or more) ALINK and/or EARC systems are used to eliminate all of the cloaked cells.
  • the invention provides a cell of the invention or a composition of the invention for use in treating a disease or condition in a subject in need thereof.
  • disease or condition is blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., Type 1 or Type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, a neurological disease, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS, an enzyme or hormone deficiency, a metabolic disorder (e.g., a lysosomal storage disorder, Galactosemia, Maple syrup urine disease, Phenylketonuria, a glycogen storage disease, a mitochondrial disorder, Friedrich's ataxia, a peroxisomal disorder, a metal metabolism disorder, or an organic academia), an autoimmune disease (e.g., Psoriasis, Systemic Lupus Erythematos), atopic a
  • the invention provides a cell of the invention or a composition of the invention for use in providing a local immunosuppression at a transplant site in an allogeneic host.
  • the cell is comprises two of the set of transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1 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-L1, HLA-G (H2-M3) and
  • the cell comprises three of the set of transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), and Cd47; PD-L1, HLA-G (H2-M3), and Cd200; PD-L1, HLA-G (H2-M3), and FASLG (FasL); PD-L1, HLA-G (H2-M3), and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), and Mfge8; PD-L1, HLA-G (H2-M3), and Serpin B9 (Spi6); PD-L1, Cd47, and Cd200; PD-L1, Cd47, and FASLG (FasL), PD-L1, HLA
  • the cell comprises four of the set of transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21 b), 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 (Ccl21 b); 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
  • the cell comprises five of the set of transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21 b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), Cd47, Cd200, and FASLG (FasL); PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), and Ccl21 (Cpi6) (e.g., PD-
  • the cell comprises seven of the set of transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) (e.g., PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Mfge8, and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (Fa
  • the cell comprises all eight of the set of transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).
  • the cell comprises one or more (e.g., one, two, three, four, five, six, or all seven) of the set of transgenes HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).
  • HLA-G H2-M3
  • Cd47 Cd200
  • FASLG FasL
  • Ccl21 Ccl21b
  • Mfge8 Serpin B9
  • the cell comprises one or more (e.g., one, two, three, four, five, six, or all seven) of the set of transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).
  • the cell comprises one or more (e.g., one, two, three, four, five, or all six) of the set of transgenes HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).
  • the cell is not modified to express PD-L1.
  • the cell is not modified to express FasL.
  • the cell is not modified to express TGF- ⁇ . 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 an NF ⁇ b decoy receptor.
  • 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 directed to IL-12.
  • the term “about” refers to a value that is no more than 10% above or below the value being described.
  • the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.
  • the term “activated leukocyte” refers to the state of a leukocyte (e.g., a granulocyte, such as a neutrophil, eosinophil, or basophil; a monocyte, or a lymphocyte, such as a B or T cell) caused by response to a perceived insult.
  • a leukocyte e.g., a granulocyte, such as a neutrophil, eosinophil, or basophil; a monocyte, or a lymphocyte, such as a B or T cell
  • leukocytes 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
  • Leukocytes can also be activated in vitro using anti-CD3/CD28 beads or other methods employed by those of skill 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)).
  • allogeneic means cells, tissue, DNA, or factors taken or derived from a different subject of the same species.
  • stem cell refers to a cell that can differentiate into one or more specialized cells and has the capacity for self-renewal.
  • Stem cells include pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and multipotent stem cells, such as cord blood stem cells, mesenchymal stromal cells and adult stem cells, which are found in various tissues.
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • multipotent stem cells such as cord blood stem cells, mesenchymal stromal cells and adult stem cells, which are found in various tissues.
  • stem cell also includes cells amenable for genome editing, cells that can serve as a source of a therapeutic cell type (e.g., cells that can be directed to differentiate into a lineage restricted or terminally differentiated cell that is used for cell therapy, or cells of a desired target tissue), and cells with “artificial” cell acquired stem cell properties (e.g., pluripotency or multipotency or self-renewal).
  • a therapeutic cell type e.g., cells that can be directed to differentiate into a lineage restricted or terminally differentiated cell that is used for cell therapy, or cells of a desired target tissue
  • cells with “artificial” cell acquired stem cell properties e.g., pluripotency or multipotency or self-renewal.
  • embryonic stem cell and “ES cell” refer to an embryo-derived totipotent or pluripotent stem cell, derived from the inner cell mass of a blastocyst that can be maintained in an in vitro culture under suitable conditions.
  • ES cells are capable of differentiating into cells of any of the three vertebrate germ layers, e.g., the endoderm, the ectoderm, or the mesoderm.
  • ES cells are also characterized by their ability propagate indefinitely under suitable in vitro culture conditions. See, for example, Thomson et al., Science 282:1145 (1998).
  • iPS cell As used herein, the terms “induced pluripotent stem cell,” “iPS cell,” and “iPSC” refer to a pluripotent stem cell that can be derived directly from a differentiated somatic cell.
  • Human iPS cells can be generated by introducing specific sets of reprogramming factors into a non-pluripotent cell that can include, for example, Oct3/4, Sox family transcription factors (e.g., Sox1, Sox2, Sox3, Sox15), Myc family transcription factors (e.g., c-Myc, 1-Myc, n-Myc), Kruppel-like family (KLF) transcription factors (e.g., KLF1, KLF2, KLF4, KLF5), and/or related transcription factors, such as NANOG, LIN28, and/or Glis1.
  • Sox family transcription factors e.g., Sox1, Sox2, Sox3, Sox15
  • Myc family transcription factors e.g., c-Myc, 1-Myc, n-
  • Human iPS cells can also be generated, for example, by the use of miRNAs, small molecules that mimic the actions 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., the endoderm, the ectoderm, or the mesoderm.
  • Human iPS cells are also characterized by their ability propagate indefinitely under suitable in vitro culture conditions. See, for example, Takahashi and Yamanaka, Cell 126:663 (2006).
  • the term “mitigate antigen presenting cell activation and function” refers to a transgene that encodes a gene product whose function is to inhibit antigen presenting cell activation or the ability of an antigen presenting cell to promote the activation of graft attacking leukocytes (Fiorentino et al., J Immunol. 146:3444-51 (1991); Salio et al., Eur J Immunol. 29:3245-53 (1999)).
  • mitigation of antigen presenting cell activation and function refers to a decrease in APC activation and function 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 antigen presenting cell activation, such as reduced proliferation, reduced secretion of pro-inflammatory cytokines (e.g., interleukin-1 (IL-1, e.g., IL-1 ⁇ ), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF, e.g., TNF ⁇ ), interferon gamma (IFN ⁇ ), and granulocyte macrophage colony stimulating factor (GMCSF), which can be measured using an ELISA or Western Blot analysis of culture media or a patient sample, such as a blood sample), or reduced levels of cell surface markers (e.g., CD11c, CD11b, HLA molecules), or reduced
  • Antigen presenting cells include dendritic cells, B cells, and macrophages. Mast cells and neutrophils can also be induced to present antigens. Methods for determining mitigation of antigen presenting cell activation and function are known in the art. Examples of gene products that mitigate antigen presenting cell activation and function include, but are not limited to: Ccl21 (Ccl21b) and PD-L1. Such transgenes may be referred herein to “cloaking” or “cloaked” genes.
  • the term “mitigate graft attacking leukocyte activity or cytolytic function” refers to a transgene that encodes a gene product whose function is to inhibit or prevent graft attacking leukocyte activity or cytolytic function near allograft cells (MacDonald et al., J Immunol. 126:1671-5 (1981); Bongrand et al., Eur J Immunol. 13:424-9 (1983); MacDonald et al., Eur J Immunol. 9:466-70 (1979)).
  • mitigation of graft attacking leukocyte activity or cytolytic function refers to a decrease in leukocyte activity or cytolytic function of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, relative to a control (e.g., as determined using an assay for leukocyte activation, such as reduced proliferation, reduced secretion of pro-inflammatory cytokines (e.g., interleukin-1 (IL-1, e.g., IL-1, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF, e.g., TNF ⁇ ), interferon gamma (IFN ⁇ ), and granulocyte macrophage colony stimulating factor (GMCSF), which can be measured using an ELISA or Western Blot analysis of culture media or a patient sample, such as a blood sample), or reduced polarization (e.g., a reduction in the level of IL
  • Methods for determining mitigation of graft attacking leukocyte activity or cytolytic function are known in the art.
  • Examples of gene products that mitigate graft attacking leukocyte activity or cytolytic function include, but are not limited to: PD-L1, HLA-G (H2-M3), Cd39, Cd73, and Lag3.
  • Such transgenes may be referred herein to “cloaking” or “cloaked” genes.
  • the term “mitigate macrophage cytolytic function and phagocytosis of allograft cells” refers to a transgene that encodes a gene product whose function is to inhibit or prevent macrophage cytolytic function and/or phagocytosis of allograft cells (Fish et al., Toxicology. 19:127-38. (1981); Sung et al., J Biol Chem. 260:546-54 (1985); Amash et al., J Immunol. 196:3331-40 (2016)).
  • mitigation of macrophage cytolytic function and phagocytosis of allograft cells refers to a decrease in macrophage cytolytic function and/or phagocytosis of allograft cells 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 macrophage cytolytic function (e.g., by incubating macrophages with a target cell line that has been pre-coated with antibodies to a surface antigen expressed by the target cell line and measuring the number of surviving target cells with a fluorescent viability stain, or by measuring the secretion of cytolytic granules (e.g., perforin, granzymes, or other cytolytic proteins released from immune cells) released from the macrophages; or as determined using an assay for macrophage phagocytosis (e.g., culturing macrophages with fluorescent beads or a target
  • the term “induce apoptosis in graft attacking leukocytes” refers to a transgene that encodes a gene product whose function is to kill graft attacking leukocytes near allograft 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)).
  • induction of apoptosis in graft attacking leukocytes refers to an increase in apoptosis in graft attacking leukocytes 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 use of fluorescent viability stains).
  • an assay for apoptosis such as TUNEL staining, caspase staining, or Annexin-V staining, or use of fluorescent viability stains.
  • Examples of gene products that can induce apoptosis in graft attacking leukocytes include, but are not limited to: FASLG (FasL) and Infsf10. Such transgenes may be referred herein to “cloaking” or “cloaked” genes.
  • the term “mitigate local inflammatory proteins” refers to a transgene that encodes a gene product whose function is to inhibit the activity of local proteins, where the function of said proteins is to promote graft attacking leukocyte accumulation, and/or their cytolytic function (Felix et al., Nat Rev Immunol. 17:112-29 (2017)).
  • mitigation of local inflammatory proteins refers to a reduction 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 a site of inflammation (e.g., a chemokine, such as CCL2, CCL3, CCLS, CXCL1, CXCL2, and CXCL8, or a pro-inflammatory cytokine, such as IL-1 ⁇ , IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, TNF ⁇ , IFN ⁇ , or GMCSF, which can be measured using an ELISA, Western blot analysis, or other techniques known in the art for measuring secreted proteins)).
  • a chemokine such as CCL2, CCL3, CCLS, CXCL1, CXCL2, and CXCL8
  • a pro-inflammatory cytokine such
  • the term “protect against leukocyte-mediated apoptosis” refers to a transgene that encodes a gene product whose function is to inhibit any cell component that may induce apoptosis or cytolysis of an allograft cell (Abdullah et al., J Immunol. 178:3390-9 (2007)).
  • protection against leukocyte-mediated apoptosis refers to a decrease in leukocyte-mediated apoptosis of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%, relative to a control (e.g., as determined using an assay for leukocyte-mediated apoptosis (e.g., by incubating leukocytes with a target cell line that has been pre-coated with antibodies to a surface antigen expressed by the target cell line and measuring the number of surviving target cells with a fluorescent viability stain, or by measuring the secretion of cytolytic granules (e.g., perforin, granzymes, or other cytolytic proteins released from immune cells) released from the leukocyte).
  • cytolytic granules e.g., perforin, granzymes, or other cytolytic proteins released from immune cells
  • biological refers to a designed polypeptide and corresponding encoding DNA, which can be expressed as a transgene.
  • the polypeptide may 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.
  • the agonist function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 95% or 100% of the function, relative to the function of a control.
  • the antagonist function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 95% or 100% of the function, relative to the function of a control.
  • operably linked refers to a first molecule joined to a second molecule, wherein the molecules are so arranged 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.
  • a promoter is operably linked to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in a cell.
  • two portions of a transcription regulatory element are operably linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion.
  • Two transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present.
  • promoter refers to a recognition site on DNA that is bound by an RNA polymerase.
  • the polymerase drives transcription of the transgene.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to 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 that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • 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 that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:
  • X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and where Y is the total number of nucleic acids in B.
  • sequence alignment program e.g., BLAST
  • Y is the total number of nucleic acids in B.
  • the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and/or carriers, to be administered to a subject, such as a mammal, e.g., a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the subject.
  • the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response and/or other problem complications commensurate with a reasonable benefit/risk ratio.
  • wild-type refers to a genotype with the highest frequency for a particular gene in a given organism.
  • cell division locus refers to a genomic locus (or loci) whose transcription product(s) is expressed by dividing cells.
  • a CDL comprises a single locus
  • absence of CDL expression in a cell (or its derivatives) means that tumour initiation and/or formation is prohibited either because the cell(s) will be ablated in the absence of CDL expression or because proliferation of the cell(s) will be blocked or compromised in the absence of CDL expression.
  • a CDL comprises multiple loci
  • absence of expression by all or subsets of the loci in a cell (or its derivatives) means that tumour initiation and/or formation is prohibited either because the cell(s) will be ablated in the absence of CDL expression or because proliferation of the cell(s) will be blocked or compromised in the absence of CDL expression.
  • a CDL may or may not be expressed in non-dividing and/or non-proliferating cells.
  • a CDL may be endogenous to a host cell or it may be a transgene. If a CDL is a transgene, it may be from the same or different species as a host cell or it may be of synthetic origin. In an embodiment, a CDL is a single locus that is transcribed during cell division.
  • a single locus CDL is CDK1.
  • a CDL comprises two or more loci that are transcribed during cell division.
  • a multi-locus CDL comprises two MYC genes (c-Myc and N-myc) (Scognamiglio et al., 2016).
  • a multi-locus CDL comprises AURORA B and C kinases, which may have overlapping functions (Fernandez-Miranda et al., 2011). Cell division and cell proliferation are terms that may be used interchangeably herein.
  • normal rate of cell division refers to a rate of cell division and/or proliferation that is typical of a non-cancerous healthy cell.
  • a normal rate of cell division and/or proliferation may be specific to cell type. For example, it is widely accepted that the number of cells in the epidermis, intestine, lung, blood, bone marrow, thymus, testis, uterus and mammary gland is maintained by a high rate of cell division and a high rate of cell death. In contrast, the number of cells in the pancreas, kidney, cornea, prostate, bone, heart and brain is maintained by a low rate of cell division and a low rate of cell death (Pellettieri and Sanchez Alvarado, 2007).
  • inducible negative effector of proliferation refers to a genetic modification that facilitates use of CDL expression to control cell division and/or proliferation by: i) inducibly stopping or blocking CDL expression, thereby prohibiting cell division and proliferation; ii) inducibly ablating at least a portion of CDL-expressing cells (i.e., killing at least a portion of proliferating cells); or iii) inducibly slowing the rate of cell division relative to a cell's normal cell division rate, such that the rate of cell division would not be fast enough to contribute to tumor formation.
  • ablation link and “ALINK” as used herein, refer to an example of an iNEP, which comprises a transcriptional link between a CDL and a sequence encoding a negative selectable marker.
  • the ALINK modification allows a user to inducibly kill proliferating host cells comprising the ALINK or inhibit the host cell's proliferation by killing at least a portion of proliferating cells by exposing the ALINK-modified cells to an inducer of the negative selectable marker.
  • a cell modified to comprise an ALINK at a CDL may be treated with an inducer (e.g., a prodrug) of the negative selectable marker in order to ablate proliferating cells or to inhibit cell proliferation by killing at least a portion of proliferating cells.
  • exogenous activator of regulation of CDL and “EARC” as used herein, refer to an example of an iNEP, which comprises a mechanism or system that facilitates exogenous alteration of non-coding or coding DNA transcription or corresponding translation via an activator.
  • An EARC modification allows a user to inducibly stop or inhibit division of cells comprising the EARC by removing from the EARC-modified cells an inducer that permits transcription and/or translation of the EARC-modified CDL.
  • an inducible activator-based gene expression system may be operably linked to a CDL and used to exogenously control expression of a CDL or CDL translation, such that the presence of a drug inducible activator and corresponding inducer drug are required for CDL transcription and/or translation.
  • a drug inducible activator and corresponding inducer drug are required for CDL transcription and/or translation.
  • cell division and/or proliferation would be stopped or inhibited (e.g., slowed to a normal cell division rate).
  • the CDL Cdk1/CDK1 may be modified to comprise a dox-bridge, such that expression of Cdk1/CDK1 and cell division and proliferation are only possible in the presence of an inducer (e.g., doxycycline).
  • proliferation antagonist system refers to a natural or engineered compound(s) whose presence inhibits (completely or partially) proliferation of a cell.
  • dox-bridge refers to a mechanism for separating activity of a promoter from a target transcribed region by expressing rtTA (Gossen et al., 1995) by the endogenous or exogenous promoter and rendering the transcription of target region under the control of TRE.
  • rtTA refers to the reverse tetracycline transactivator elements of the tetracycline inducible system (Gossen et al., 1995)
  • TRE refers to a promoter consisting of TetO operator sequences upstream of a minimal promoter.
  • the rtTA sequence may be inserted in the same transcriptional unit as the CDL or in a different location of the genome, so long as the transcriptional expression's permissive or non-permissive status of the target region is controlled by doxycycline.
  • a dox-bridge is an example of an EARC.
  • Fail-safe cell refers to a cell that contains one or more homozygous, heterozygous, hemizygous or compound heterozygous ALINKs or EARCs in one or more CDLs (e.g., at least two, three, four, or five CDLs). Fail-safe cells may contain either ALINKs or EARCs or both ALINK and EARC modifications (e.g., ALINK and EARC modifications in different CDLs or in a single CDL).
  • the term “fail-safe” refers to a property of a cell that is unlikely to exhibit uncontrolled (e.g., tumorigenic) proliferation.
  • a cell can be considered “fail safe” when cell proliferation is under the control of a negative regulator or inducer, and the possibility of the cell losing the activity of the system that controls proliferation due to genetic mutation is low.
  • the fail-safe volume will depend on the number of ALINKs and the number of ALINK-targeted CDLs (e.g., a cell with homozygous modifications of two different CDLs has a higher fail safe volume (e.g., it is less likely to lose all systems that control proliferation through genetic mutation) than a cell with a heterozygous modification of a single CDL).
  • the fail-safe property is further described in Table 3.
  • FIGS. 1A-1D depict representative images showing the expression of cloaking proteins (Cd200 ( FIG. 1A ), FasL ( FIG. 1B ), H2-M3 ( FIG. 1C ) and Cd47 ( FIG. 1D )) in C57BL/6 mouse embryonic stem cell line C2 using immunohistochemistry.
  • FIGS. 2A-2E are flow cytometry plots showing T-cell activation using splenocytes ( FIG. 2A ), wt B16 melanoma cells ( FIG. 2B ), cloaked B16 melanoma cells ( FIG. 2C ), wt ES cells ( FIG. 2D ), and cloaked ES cells ( FIG. 2E ) in a mixed lymphocyte reaction.
  • FIGS. 3A-3B are schematics and images showing that cloaked ( FIG. 3B ) B16F10 cancer cells in an allogenic model are protected from rejection compared to their WT counterparts ( FIG. 3A ).
  • FIG. 4 is a schematic showing that cloaked embryonic stem cells form tumors in isogenic B6 mouse recipients (upper panel) and in FVB allogenic recipients (lower panel).
  • FIGS. 5A-5C are a series of photographs depicting allogenic mice bearing teratomas formed from subcutaneous injection of cloaked C57BL/6 ES cells. Red arrows indicate teratomas.
  • FIG. 5A shows teratomas in C3H mice
  • FIG. 5B shows teratomas in FVB/N mice
  • FIG. 5C shows teratomas in CD1 mice.
  • FIG. 6 is a schematic and series of images showing that animals with cloaked tissue are not immune compromised.
  • FIG. 7 is a series of images of FVB/N mice showing additional results showing that animals with cloaked tissues are not immune compromised.
  • FIGS. 8A-8H show transgene expression in clonal FailSafe containing embryonic stem cells derived from C57BL/6 mice.
  • FIG. 8A shows FasL expression
  • FIG. 8B shows Ccl21 b expression
  • FIG. 8C shows Cd200 expression
  • FIG. 8D shows Cd47 expression
  • FIG. 8E shows Mfge8 expression
  • FIG. 8F shows Spi6 expression
  • FIG. 8H shows PD-L1 expression.
  • FIG. 9 is a series of graphs depicting cloaking transgene expression in ES cell clones. Each cloaking transgene is depicted in a different color. Concentric circles represent expression level on a log 10 scale. The thick black circle represents ix expression normalized to positive controls (activated leukocytes isolated from murine lymph organs), with the next outer ring representing 10 ⁇ and 100 ⁇ expression compared to positive controls, respectively. The innermost ring is 0.1 ⁇ expression compared to positive controls. Clones NT2 and 15 (indicated with red squares) had the highest expression of the cloaking genes. These clones survived in allogenic hosts.
  • FIG. 10 is a graph depicting the expression of the cloaking transgenes among the whole genome gene expression level distribution for the whole genome of ES cells.
  • All 8 cloaking transgenes in the NT2 cell line and NT2-derived teratoma had an expression level that was among the top 5% of all genes in the ES cell genome, with 5 of the cloaking transgenes having an expression level in the top 1% of all genes in the ES cell genome.
  • the expression levels of the transgenes in the NT2 line and NT2-derived teratoma succeeded to achieve allograft tolerance.
  • FIGS. 11A-11B are photographs showing C57BL/6 derived teratomas in FVB/N mice.
  • the transgenic line, NT2 resulted in 9 teratomas out of 10 injection sites. Images were taken 3 months post injection.
  • FIG. 11B is an enlarged image of the teratoma indicated by the arrow in FIG. 11A .
  • FIGS. 12A-12B are graphs showing the teratoma tumor size in isogenic ( FIG. 12A ) and allogenic ( FIG. 12B ) mice treated with ganciclovir.
  • FIGS. 13A-13B are a series of photomicrographs showing that cloaked embryonic stem cells, injected into both isogenic ( FIG. 13A ) and allogenic ( FIG. 13B ) hosts, can differentiate into all three cell lineages.
  • FIGS. 14A-140 are photomicrographs showing the formation of all three germ layers in a teratoma formed from subcutaneous injection of cloaked ES cells into a mouse.
  • FIG. 14D shows a blood vessel, indicated by the red arrow, confirming that the tissues are well vascularized.
  • FIG. 15 is a schematic showing the construction of vectors that express target genes essential for allo-tolerance.
  • FIGS. 16A-16H are fluorescent photomicrographs showing the expression of proteins encoded by the cloaking transgenes in ES cells.
  • FIG. 16A shows the expression of PD-L1
  • FIG. 16B shows the expression of CD200
  • FIG. 16C shows the expression of CD47
  • FIG. 16D shows the expression of FasL
  • FIG. 16E shows the expression of H2-M3
  • FIG. 16F shows the expression of Ccl21
  • FIG. 16G shows the expression of Mfge8, and
  • FIG. 16H shows the expression of Spi6.
  • FIGS. 17A-17B are photomicrographs showing that cloaked ES cells have typical ES cell morphology ( FIG. 17A ) and express the ES cell marker alkaline phosphatase ( FIG. 17B ).
  • FIGS. 18A-18B are fluorescent photomicrographs showing the expression of markers of pluripotent ES cells (Oct4 ( FIG. 18A ) and SSEA1 ( FIG. 18B )) in cloaked ES cells.
  • the insets in FIGS. 18A-18B show single channel images of the fluorescent micrographs for the ES cell markers (Oct4 and SSEA) and DAPI, which labels the nucleus, to demonstrate that staining for the ES cell markers colocalizes with the cloaked cells.
  • FIG. 19 is a schematic depicting the immune processes that are inhibited by the cloaking transgenes (top) and the expression cassette (bottom) used to express the cloaking transgenes in ES cells.
  • FIG. 20 is a series of graphs depicting the effect of interferon gamma (IFN ⁇ ) on MHC levels in ES cells.
  • IFN ⁇ interferon gamma
  • IFN ⁇ increased MHC levels in wild-type ES cells and ES cells overexpressing the wild-type IFN ⁇ receptor IFN ⁇ R1, but did not increase MHC levels in ES cells overexpressing a dominant negative form of the IFN ⁇ receptor (IFN ⁇ R1 d39), indicating that IFN ⁇ R1 d39 completely inhibited the IFN ⁇ -mediated upregulation of MHCs in ES cells.
  • the genetically modified cell comprises: one or a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting and whose function is to mitigate function of the host immune system (e.g., graft attacking leukocyte and NK cell activation) or act as a defense mechanism against attacking leukocytes.
  • Immune factors like PD-L1 (Brown et al., J Immunol. 170:1257-66 (2003: Curie) et al., Nat Med. 9:562-7 (2003); Dong et al., Nat Med. 8:793-800 (2002)), CD47 ((Willingham et al., Proc Natl Aced Sci USA. 109:6662-7 (2012); Liu et al., PLoS One. 10:e0137345 (2015); Demeure et al., J Immunol.
  • PD-L1 Brown et al., J Immunol. 170:1257-66 (2003: Curie) et al., Nat Med. 9:562-7 (2003); Dong et al., Nat Med. 8:793-800 (2002)
  • CD47 (Willingham et al., Proc Natl Aced Sci USA. 109:6662-7 (2012); Liu et al., PLoS One.
  • the modified cells evade immune rejection through the simultaneous modulation of many different local immune pathways.
  • Such genetically engineered cells can be transplanted “off the shelf” into many recipients regardless of genetic background and without rejection by the recipient's immune system.
  • This immunomodulatory approach overcomes the requirement for systemic immunosuppression of the transplant recipient, which can be dangerous to the recipient.
  • an immunosuppressive agent(s) can be administered to a patient that receives the modified cells described herein, the therapy need not include the administration of an immunosuppressive agent(s).
  • This immunomodulatory approach also overcomes the costly and impractical methodology of deriving patient-specific iPS cells, manipulating regulatory cells, or inducing chimerism through hematopoietic cell transplantation (HCT).
  • Cells can be genetically modified to express a set of transgenes encoding gene products that are cytoplasmic, membrane bound, or local acting, and whose function is to mitigate immune function (e.g., graft attacking leukocyte and NK cell activation) or to act as a defense mechanism against the immune response (e.g., attacking leukocytes).
  • the set of transgenes may be selected from the genes having a role in the immune modulatory pathways described above. Such genes include, but are not limited to those provided in Table 1.
  • C-C motif chemokine ligand 21(Ccl21) is expressed by local lymph nodes where it acts to attract activated antigen presenting cells (APCs). This key function offers an opportunity to “reverse” the migration of APCs by overexpressing this gene on grafted cells. Indeed, some melanomas express Ccl21 and recruit CCR7 + cells that, in turn, can reorganize portions of their tumor stroma as “self”. This leads to a stromal reconstruction that directs the recruitment and maintenance of Cd4 + Tregs (Zindl et al., Science. 328:697-8 (2010)).
  • Ccl21 b is the mouse ortholog of human Ccl21.
  • amino acid sequences of mouse and human Ccl21 are:
  • Cd47 in umbilical cord blood can promote the development of hyporesponsive T-cells (Avice et al., J Immunol. 167:2459-68 (2001)). Erythrocytes also up-regulate Cd47 to avoid dendritic cell activation due to their lack of “self” presentation (van den Berg et al., Immunity. 43:622-4 (2015)). More recently, it was shown that expression of human Cd47 increases engraftment in a mouse model of pig-to-human hematopoietic cell transplantation (Tena et al., Am J Transplant. 14:2713-22 (2014)).
  • amino acid sequences of mouse and human Cd47 are:
  • Cd200 is also as an important immunoregulatory molecule; increased expression can reduce the severity of allograft rejection, autoimmunity, and allergic disease (Gorczynski et al., J Immunol. 172:7744-9 (2004)). It has been shown that, in vitro, APC expression of Cd200 suppresses production of interferon gamma (IFN- ⁇ ) and cytolytic granules by activated Cd8+ T-cells (Misstear et al., J Virol. 86:6246-57 (2012)). Most interestingly, overexpression of Cd200 increases the survival of skin and cardiac allografts in mice by promoting of Foxp3+ Treg cells (Gorczynski et al., Transplantation. 98:1271-8 (2014)).
  • IFN- ⁇ interferon gamma
  • Cd8+ T-cells cytolytic granules by activated Cd8+ T-cells
  • amino acid sequences of mouse and human Cd200 are:
  • Spi6 is an endogenous inhibitor of the cytotoxic effector molecule granzyme B released by activated Cd8+ T-cells (Sun et al., J Biol Chem. 272:15434-41 (1997)). Some data shows that Mesenchymal Stem Cells (MSCs) escape 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 themselves being killed through contact mediated cytotoxicity is mediated by Spi6 (Lovo et al., J Immunol. 188:1057-63 (2012)). Spi6 is also known as Serpin B9.
  • amino acid sequences of mouse Spi6 and the human counterpart, Serpin B9 are:
  • Mouse Spi6 (SEQ ID NO: 7) MNTLSEGNGTFAIHLLKMLCQSNPSKNVCYSPASISSALAMVLLGAK GQTAVQISQALGLNKEEGIHQGFQLLLRKLNKPDRKYSLRVANRLFA DKTCEVLQTFKESSLHFYDSEMEQLSFAEEAEVSRQHINTWVSKQTE GKIPELLSGGSVDSETRLVLINALYFKGKWHQPFNKEYTMDMPFKIN KDEKRPVQMMCREDTYNLAYVKEVQAQVLVMPYEGMELSLVVLLPDE GVDLSKVENNLTFEKLTAWMEADFMKSTDVEVFLPKFKLQEDYDMES LFQRLGVVDVFQEDKADLSGMSPERNLCVSKFVHQSVVEINEEGTEA AAASAIIEFCCASSVPTFCADHPFLFFIRHNKANSILFCGRFSSP Human Serpin B9 (SEQ ID NO: 8) METLSNASGTFAIRLLKILCQDNP
  • FasL Activated, cytotoxic, Cd8+ can kill target cells by expression of FasL, which binds to the FAS receptor and activates a caspase-mediated apoptosis in targeted cells.
  • FasL binds to the FAS receptor and activates a caspase-mediated apoptosis in targeted cells.
  • many tumors have developed a “counterattack” by upregulating FasL on their surface (Chen et al., J Immunol. 171:1183-91 (2003)).
  • Selective expression of FasL in the vasculature of human and mouse solid tumors has been associated with scarce Cd8+ T-cell infiltration and a predominance of FoxP3+ Treg cells (Motz et al. Nat Med. 20:607-15 (2014)).
  • FasL is the mouse ortholog of human FASLG.
  • PD-L1 is a critical immune modulatory 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)).
  • amino acid sequences of mouse and human PD-L1 are:
  • Mouse PD-L1 (SEQ ID NO: 11) MRIFAGIIFTACCHLLRAFTITAPKDLYVVEYGSNVTMECRFPVERE LDLLALVVYWEKEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLK GNAALQITDVKLQDAGVYCCIISYGGADYKRITLKVNAPYRKINQRI SVDPATSEHELICQAEGYPEAEVIVVTNSDHQPVSGKRSVTTSRTEG MLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPP QNRTHWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKN RNDTQFEET Human PDL1 (CD274) (SEQ ID NO: 12) MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQ LDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRA
  • Inflammatory environments like those induced by allograft transplants, attracts macrophages and inflammatory monocytes, among many other innate immune cells.
  • the milk fat globule epidermal growth factor-8 (Mfge-8) is expressed by many murine tumours (Neutzner et al., Cancer Res. 67:6777-85 (2007)) and has been shown to contribute to local immune suppression by polarizing incoming monocytes to suppressive, M2-like macrophages (Soki et al., J Biol Chem. 289:24560-72 (2014)).
  • amino acid sequences of mouse and human MFGE-8 are:
  • NK cells can kill targets cells that lack MHC class I molecules, as well as other cells within an inflammatory setting.
  • H2-M3 the murine homologue of human HLA-G has recently been shown to have a regulatory effect on NK cells, licensing them to ignore cells that lack “self molecules” (Andrews et al., Nat Immunol. 13:1171-7 (2012)). This is thought to be achieved by binding of HLA-G, immunosuppressive receptors on both NK and T-cells (Carosella et al., Adv Immunol. 127:33-144 (2015)).
  • H2-M3 is the mouse ortholog of human HLA-G.
  • amino acid sequences of mouse H2-M3 and the human counterpart, HLA-G are:
  • a set of transgenes that includes one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21 b, Mfge8, and Spi6A can be expressed in cells.
  • the cells may be, for example, stem cells or a cell that is amenable to genome editing, such as a cell that can be used for therapy and/or differentiated into a therapeutic cell type.
  • the stem cells may be, for example, embryonic stem (ES) cells or induced pluripotent stem (iPS) cells.
  • the set of transgenes may comprise 1, 2, 3, 4, 5, 6, 7, or all 8 of these genes or may comprise 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 cell may be further genetically modified to express one or more of TGF- ⁇ , Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and/or IFN ⁇ R1 d39.
  • the TGF- ⁇ transgene may be modified to express the gene product in a membrane-bound form (i.e., such that the gene product is expressed on the cell surface of the allograft), using methods known to those skilled in the art.
  • a method for localizing TGF- ⁇ to the membrane is to co-express TGF- ⁇ with an additional transgene encoding the LRRC32 protein or any other polypeptide that results in localization of TGF- ⁇ to the cell membrane. This protein anchors TGF- ⁇ to the membrane.
  • the amino acid sequence of IFN ⁇ R1 d39 is:
  • the genes may be human genes or murine genes.
  • the gene is of the same species as the recipient of the allograft recipient in which the cell is to be transplanted.
  • the gene is of any species in which the function of the gene is conserved or in which a designed biologic has the agonist function of the endogenous counterpart. Methods for introducing and expressing these transgenes in cells are described herein and are also known to those skilled in the art. Cells expressing these transgenes may be referred to as “cloaked” due to their ability to evade allorejection without systemic immunosuppression and without the need for immunosuppressive drugs.
  • populations of cells derived from the above-described cloaked cells can also be used to produce a local immunosuppression when transplanted at a transplant site of an allogeneic recipient.
  • the cells can first be modified to be fail-safe cells.
  • Fail-safe cells use cell division loci (CDLs) to control cell proliferation in animal cells.
  • CDLs are loci whose transcription product(s) are expressed during cell division.
  • CDLs may be genetically modified, as described herein, to comprise a negative selectable marker and/or an inducible activator-based gene expression system, which allows a user to permit, ablate, and/or inhibit proliferation of the genetically modified cell(s) by adding or removing an appropriate inducer.
  • Methods for making and using fail-safe cells are described, for example, in WO 2016/141480, the entire teachings of which are incorporated herein by reference.
  • a cell may be made fail-safe first and then cloaked afterwards.
  • a cell may be cloaked first and then made fail-safe afterwards.
  • the cell may be a vertebrate cell, for example, a mammalian cell, such as a human cell or a mouse cell.
  • the cell may also be a vertebrate stem cell, for example, a mammalian stem cell, such as a human stem cell or a mouse stem cell.
  • the cell or stem cell is amenable to genetic modification.
  • the cell or stem cell is deemed by a user to have therapeutic value, meaning that the cell or stem cell may be used to treat a disease, disorder, defect or injury in a subject in need of treatment for same.
  • the cell is a stem cell or progenitor cell (e.g., iPSC, embryonic stem cell, hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem or progenitor cells, germline stem cells, lung stem or progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, or neural stem or progenitor cells).
  • the stem cells are adult stem cells (e.g., somatic stem cells or tissue specific stem cells).
  • the stem or progenitor cell is capable of being differentiated (e.g., the stem cell is totipotent, pluripotent, or multipotent).
  • the cell is isolated from embryonic or neonatal tissue.
  • the cell is a fibroblast, monocytic precursor, B cell, exocrine cell, pancreatic progenitor, endocrine progenitor, hepatoblast, myoblast, preadipocyte, progenitor cell, hepatocyte, chondrocyte, smooth muscle cell, K562 human erythroid leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipose cell, dendritic cells, or natural killer cell.
  • the cell is manipulated (e.g., converted or differentiated) into a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or brown adipocyte.
  • the cell is a muscle cell (e.g., skeletal, smooth, or cardiac muscle cell), erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell (e.g., red blood cell, white blood cell, or platelet), endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or white or brown adipocyte.
  • muscle cell e.g., skeletal, smooth, or cardiac muscle cell
  • erythroid-megakaryocytic cell eosinophil
  • iPS cell eosinophil
  • macrophage macrophage
  • T cell islet beta-cell
  • neuron e.g., cardiomyocyte
  • blood cell e.g., red blood cell, white blood cell,
  • the cell is a hormone-secreting cell (e.g., a cell that secretes insulin, oxytocin, endorphin, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormone, bombesin, cholecystokinin, testosterone, estrogen, or progesterone, renin, ghrelin, amylin, or pancreatic polypeptide), an epidermal keratinocyte, an epithelial cell (e.g., an exocrine secretory epithelial cell, a thyroid epithelial cell, a keratinizing epithelial cell, a gall bladder epithelial cell, or a surface epithelial cell of the cornea, tongue, oral cavity, esophagus, anal canal, distal urethra, or vagina), a kidney cell, a germ cell, a skeletal joint synovium cell, a periostea cell,
  • the cell is a somatic cell.
  • the cells are derived from skin or other organs, e.g., heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.
  • the cells can be from humans or other mammals (e.g., rodent, non-human primate, bovine, or porcine cells). It is contemplated herein that cloaked cells may be of use in cell-based therapies wherein it may be desirable to evade allorejection at a localized transplant site.
  • the cloaked cells described herein survive in a host without stimulating the host immune response for one week or more (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 more, e.g., for the life of the cell and/or its progeny).
  • the cells maintain expression of the cloaking transgenes for as long as they survive in the host (e.g., if cloaking transgenes are no longer expressed, the cloaked cells may be removed by the host's immune system).
  • the cloaked cells further express a transgene encoding a protein that allows the cloaked cells to be detected in vivo (e.g., a fluorescent protein, such as GFP or other detectable marker).
  • the combination of cloaked and fail-safe cells may be of use in cell-based therapies wherein it may be desirable to evade allorejection at a localized transplant site, while also being able to eliminate cells exhibiting undesirable growth rates, irrespective of whether such cells are generated before or after grafting the cells into a host.
  • the combined cloaking and fail-safe technologies allows for localized immunoprotection while addressing the risk that the recipient will develop a malignancy because the cells are providing local immunosuppression.
  • compositions and methods described herein can be used to reduce rejection of allogenic cells through expression of cloaking transgenes.
  • a wide array of methods has been established for the delivery of proteins to mammalian cells and for the stable expression of genes encoding proteins in mammalian cells, which can be used to produce the cloaked cells described herein.
  • One platform that can be used to achieve therapeutically effective expression of cloaking proteins or therapeutic agents in mammalian cells is via the stable expression of a gene encoding a cloaking protein or therapeutic agent (e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell, or by episomal concatemer formation in the nucleus of a mammalian cell).
  • the gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein.
  • genes can be incorporated into a vector.
  • Vectors can be introduced into a cell by a variety of methods, including transformation, transfection, transduction, direct uptake, projectile bombardment, and by encapsulation of the vector in a liposomes.
  • suitable methods of 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 each of which are incorporated herein by reference.
  • Cloaking proteins or therapeutic agents can also be introduced into a mammalian cell by targeting vectors containing portions of a gene encoding a cloaking protein or therapeutic agent to cell membrane phospholipids.
  • vectors can be targeted to the phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to a VSV-G protein, a viral protein with affinity for all cell membrane phospholipids.
  • VSV-G protein a viral protein with affinity for all cell membrane phospholipids.
  • RNA polymerase Recognition and binding of the polynucleotide encoding a cloaking protein or therapeutic agent by mammalian RNA polymerase is important for gene expression.
  • sequence elements within the polynucleotide that exhibit a high affinity for transcription factors that recruit RNA polymerase and promote the assembly of the transcription complex at the transcription initiation site.
  • sequence elements include, e.g., a mammalian promoter, the sequence of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase.
  • Polynucleotides suitable for use in the compositions and methods described herein also include those that encode a cloaking protein or therapeutic agent downstream of a mammalian promoter.
  • Promoters that are useful for the expression of a cloaking protein or therapeutic agent in mammalian cells include constitutive promoters.
  • Constitutive promoters include the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1 ⁇ promoter, and the PGK promoter.
  • CMV cytomegalovirus
  • EF1 ⁇ promoter the EF1 ⁇ promoter
  • PGK promoter PGK promoter
  • promoters derived from viral genomes can also be used for the stable expression of these agents in mammalian cells.
  • adenovirus late promoter examples include 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 the Rous sarcoma virus (RSV) promoter.
  • vaccinia virus 7.5K promoter vaccinia virus 7.5K promoter
  • SV40 promoter tk promoter of HSV
  • MMTV mouse mammary tumor virus
  • LTR promoter of HIV promoter of moloney virus
  • EBV Epstein barr virus
  • RSV Rous sarcoma virus
  • the transcription of this polynucleotide can be induced by methods known in the art.
  • expression can be induced by exposing the mammalian cell to an external chemical reagent, such as an agent that modulates the binding of a transcription factor and/or RNA polymerase to the mammalian promoter and thus regulates gene expression.
  • the chemical reagent can serve to facilitate the binding of RNA polymerase and/or transcription factors to the mammalian promoter, e.g., by removing a repressor protein that has bound the promoter.
  • the chemical reagent can serve to enhance the affinity of the mammalian promoter for RNA polymerase and/or transcription factors such that the rate of transcription of the gene located downstream of the promoter is increased in the presence of the chemical reagent.
  • Examples of chemical reagents that potentiate polynucleotide transcription by the above mechanisms include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, Calif.) and can be administered to a mammalian cell in order to promote gene expression according to established protocols.
  • DNA sequence elements that may 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 a conformational change in the polynucleotide containing the gene of interest such that the DNA adopts a three-dimensional orientation that is favorable for binding of transcription factors and RNA polymerase at the transcription initiation site.
  • polynucleotides for use in the compositions and methods described herein include those that encode a cloaking protein or therapeutic agent and additionally include a mammalian enhancer sequence.
  • Enhancers for use in the compositions and methods described herein also include those that are derived from the genetic material of a virus capable of infecting a eukaryotic cell. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • Enhancer sequences that induce activation of eukaryotic gene transcription are disclosed in Yaniv, et al., Nature 297:17 (1982).
  • An enhancer may be spliced into a vector containing a polynucleotide encoding a cloaking protein or therapeutic agent, for example, at a position 5′ or 3′ to this gene. In a preferred orientation, the enhancer is positioned at the 5′ side of the promoter, which in turn is located 5′ relative to the polynucleotide encoding a cloaking protein or therapeutic agent.
  • the nucleic acid vectors described herein may include a Woodchuck Posttranscriptional Regulatory Element (WPRE).
  • WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and/or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cell.
  • the addition of the WPRE to a vector can result in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo.
  • the nucleic acid vectors for use in the compositions and methods described herein include a reporter sequence, which can be useful in verifying gene expression, for example, in specific cells and tissues.
  • Reporter sequences that may be provided in a transgene include DNA sequences encoding ⁇ -lactamase, ⁇ -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.
  • the reporter sequences When associated with regulatory elements which drive their expression, the reporter sequences provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunohistochemistry for example, where the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for ⁇ -galactosidase activity. Where the transgene is green fluorescent protein or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.
  • a transgene such as a cloaking transgene or a therapeutic transgene described herein
  • a target cell e.g., a mammalian cell
  • electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest.
  • mammalian cells such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids.
  • Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference.
  • a similar technique, NucleofectionTM utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell.
  • NucleofectionTM and protocols useful for performing this technique are described in detail, e.g., in Distler et al., Experimental Dermatology 14:315 (2005), as well as in US 2010/0317114, the disclosures of each of which are incorporated herein by reference.
  • Additional techniques useful for the transfection of target cells include the squeeze-poration methodology. This technique induces the rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores that form in response to the applied stress. This technology is advantageous in that a vector is not required for delivery of nucleic acids into a cell, such as a human target cell. Squeeze-poration is described in detail, e.g., in Sharei et al., Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.
  • Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for instance, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for instance, in U.S. Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference.
  • Similar techniques that exploit ionic interactions with the cell membrane to provoke the uptake of foreign nucleic acids include contacting a cell with a cationic polymer-nucleic acid complex.
  • exemplary cationic molecules that associate with polynucleotides so as to impart a positive charge favorable for interaction with the cell membrane include activated dendrimers (described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference) polyethylenimine, and diethylaminoethyl (DEAE)-dextran, the use of which as a transfection agent is described in detail, for instance, in Gulick et al., Current Protocols in Molecular Biology 40:1:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference.
  • activated dendrimers described, e.g., in Dennig, Topics in Current Chemistry 228:227 (2003), the disclosure of which is incorporated herein by reference
  • Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids. This technology is described in detail, for instance, in US 2010/0227406, the disclosure of which is incorporated herein by reference.
  • laserfection also called optical transfection
  • Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, also called optical transfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane.
  • the bioactivity of this technique is similar to, and in some cases found superior to, electroporation.
  • 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 a substrate. DNA containing the gene, intended for intracellular delivery, is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. Cells that are impaled by nanostructures can express the delivered gene(s).
  • An example of this technique is described in Shalek et al., PNAS 107: 1870 (2010), the disclosure of which is incorporated herein by reference.
  • Magnetofection can also be used to deliver nucleic acids to target cells.
  • the magnetofection principle is to associate nucleic acids with cationic magnetic nanoparticles.
  • the magnetic nanoparticles are made of iron oxide, which is fully biodegradable, and coated with specific cationic proprietary molecules varying upon the applications.
  • Their association with the gene vectors (DNA, siRNA, viral vector, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interaction.
  • the magnetic particles are then concentrated on the target cells by the influence of an external magnetic field generated by magnets. This technique is described in detail in Scherer et al., Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.
  • sonoporation a technique that involves the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.
  • Microvesicles represent another potential vehicle that can be used to modify the genome of a target cell according to the methods described herein. For instance, microvesicles that have been induced by the co-overexpression of the glycoprotein VSV-G with, e.g., a genome-modifying protein, such as a nuclease, can be used to efficiently deliver proteins into a cell that subsequently catalyzes the site-specific cleavage of an endogenous polynucleotide sequence so as to prepare the genome of the cell for the covalent incorporation of a polynucleotide of interest, such as a gene or regulatory sequence.
  • a genome-modifying protein such as a nuclease
  • vesicles also referred to as Gesicles
  • Gesicles for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract].
  • Methylation changes in early embryonic genes in cancer [abstract], in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No. 122.
  • stable expression of an exogenous gene in a mammalian cell can be achieved by integration of the polynucleotide containing the gene into the nuclear genome of the mammalian cell.
  • a variety of vectors for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of a mammalian cell have been developed. Examples of expression vectors are disclosed in, e.g., WO 1994/011026 and are incorporated herein by reference.
  • Expression vectors for use in the compositions and methods described herein contain a cloaking transgene or therapeutic transgene, as well as, e.g., additional sequence elements used for the expression of these agents and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • Certain vectors that can be used for the expression of cloaking transgenes or therapeutic transgenes include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • Other useful vectors for expression of cloaking transgenes or therapeutic transgenes contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription.
  • sequence elements include, e.g., 5′ and 3′ untranslated regions and a polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • Viral genomes provide a rich source of vectors that can be used for the efficient delivery of a gene of interest into the genome of a target cell (e.g., a mammalian cell, such as a human cell).
  • 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 typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration.
  • viral vectors examples include a retrovirus (e.g., Retroviridae family viral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
  • RNA viruses such as picornavirus and alphavirus
  • double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox).
  • herpesvirus e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus
  • poxvirus e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox
  • Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example.
  • retroviruses examples include: avian leukosis-sarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)).
  • murine leukemia viruses include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses.
  • vectors are described, for example, U.S. Pat. No. 5,801,030, the disclosure of which is incorporated herein by reference as it pertains to viral vectors for use in gene therapy.
  • cloaking transgenes or therapeutic transgenes described herein are incorporated into rAAV vectors and/or virions in order to facilitate their introduction into a cell.
  • rAAV vectors useful in the compositions and methods described herein are recombinant nucleic acid constructs that include (1) a promoter, (2) a heterologous sequence to be expressed (e.g., a cloaking transgene or therapeutic transgene described herein), and (3) viral sequences that facilitate integration and expression of the heterologous genes.
  • the viral sequences may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion.
  • Such rAAV vectors may also contain marker or reporter genes.
  • Useful rAAV vectors have one or more of the AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences.
  • the AAV ITRs may be of any serotype suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al., J. Biomed. Sci. 7:279 (2000), and Monahan and Samulski, Gene Delivery 7:24 (2000), the disclosures of each of which are incorporated herein by reference as they pertain to AAV vectors for gene delivery.
  • transgenes and vectors described herein can be incorporated into a rAAV virion in order to facilitate introduction of the polynucleotide or vector into a cell.
  • the capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV cap gene.
  • the cap gene encodes three viral coat proteins, VP1, VP2 and VP3, which are required for virion assembly.
  • the construction of rAAV virions has been described, for instance, in U.S. Pat. Nos.
  • rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAV serotypes including AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43, and rh74. Construction and use of AAV vectors and AAV proteins of different serotypes are described, for instance, 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.
  • pseudotyped rAAV vectors include AAV vectors of a given serotype (e.g., AAV9) pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.).
  • AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described, for instance, 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).
  • AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions.
  • suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
  • the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol. 74:8635 (2000).
  • Other rAAV virions that can be used in methods described herein include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat. Genet., 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).
  • Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by 5′ and 3′ excision sites. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon.
  • transposase This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In some instances, these excision sites may be terminal repeats or inverted terminal repeats.
  • the gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of similar excision sites that exist within the nuclear genome of the cell. This allows the gene of interest to be inserted into the cleaved nuclear DNA at the complementary excision sites, and subsequent covalent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the mammalian cell genome completes the incorporation process.
  • the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed to an RNA product and then reverse-transcribed to DNA before incorporation in the mammalian cell genome.
  • exemplary transposon systems are the piggybac transposon (described in detail in, e.g., WO 2010/085699) and the sleeping beauty transposon (described in detail in, e.g., US 2005/0112764), the disclosures of each of which are incorporated herein by reference as they pertain to transposons for use in gene delivery to a cell of interest.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • Cas9 Cas9 nuclease
  • Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site.
  • highly site-specific cas9-mediated DNA cleavage can be engendered in a foreign polynucleotide because the interaction that brings cas9 within close proximity of the target DNA molecule is governed by RNA:DNA hybridization.
  • RNA:DNA hybridization RNA:DNA hybridization
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • ZFNs and TALENs in genome editing applications is described, e.g., in Urnov et al., Nature Reviews Genetics 11:636 (2010); and in Joung et al., Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosure of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.
  • Additional genome editing techniques that can be used to incorporate polynucleotides encoding target genes into the genome of a target cell include the use of ARCUSTM meganucleases that can be rationally designed so as to site-specifically cleave genomic DNA.
  • the use of these enzymes for the incorporation of genes encoding target genes into the genome of a mammalian cell is advantageous in view of the defined structure-activity relationships that have been established for such enzymes.
  • Single chain meganucleases can be modified at certain amino acid positions in order to create nucleases that selectively cleave DNA at desired locations, enabling the site-specific incorporation of a target gene into the nuclear DNA of a target cell.
  • These single-chain nucleases have been described extensively in, for example, U.S. Pat. Nos. 8,021,867 and 8,445,251, the disclosures of each of which are incorporated herein by reference as they pertain to compositions and methods for genome editing.
  • the cloaking transgenes described herein are expressed in an amount sufficient to produce a cloaking effect (e.g., in an amount sufficient to prevent rejection when injected into a subject, e.g., a mammalian subject, such as a mouse, rat, or human).
  • Transgene expression can be considered to produce a cloaking effect if subcutaneous injection of cloaked cells generates a teratoma that is not removed by the subject's immune system.
  • the cloaking transgenes are also expressed at a level that is sufficient to promote production of the proteins encoded by said transgenes. Protein production can be detected using routine methods known to those of skill in the art (e.g., immunohistochemistry, Western Blot analysis, or other methods that allow for visualization or proteins).
  • the expression of the cloaking transgenes is such that all 8 proteins encoded by the cloaking transgenes (PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21 b, Mfge8, and Spi6) can be detected in cloaked cells (e.g., detected by immunohistochemistry using antibodies directed against the proteins encoded by the cloaking transgenes).
  • cloaking transgenes are expressed at similar levels in cloaked cells to levels of endogenous gene expression in activated leukocytes, such as T cells (e.g., activated leukocytes from the same species, such as an activated leukocyte isolated from a lymph organ, for example expression in a cloaked mouse cell is similar to expression in an activated leukocyte isolated from a murine lymphoid organ).
  • activated leukocytes such as T cells
  • activated leukocytes from the same species such as an activated leukocyte isolated from a lymph organ, for example expression in a cloaked mouse cell is similar to expression in an activated leukocyte isolated from a murine lymphoid organ.
  • the expression of one or more cloaking transgenes is greater than or equal to expression of the endogenous gene in activated leukocytes (e.g., T cells) from the same species (e.g., expression level of the cloaking transgene is equal to the level of expression of the endogenous gene in activated leukocytes, or is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold or more higher than the level of expression of the endogenous gene in activated leukocytes).
  • all 8 cloaking transgenes are expressed at a level that is greater than or equal to the expression level of the endogenous gene in an activated leukocyte from the same species.
  • Activated leukocytes can be isolated from lymphoid organs, or leukocytes, such as T cells, can activated in vitro using anti-CD3/CD28 beads or other methods employed by those of skill in the art (see, e.g., Frauwith and Thompson, J.Clin Invest 109:295-299 (2002); and Trickett and Kwan, J Immunol Methods 275:251-255 (2003)).
  • Transgene expression in cloaked cells can also be compared to gene expression levels reported in profiling studies of activated T cells (see, e.g., Palacios et al., PLOSone 2:e1222 (2007)).
  • cloaking transgene expression is compared to expression of the endogenous gene in a wild-type version of the cell (e.g., a stem cell, e.g., an embryonic stem cell from the same species as the cloaked cell).
  • the expression of one or more cloaking transgenes is at least 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 cloaked cells compared to expression of the endogenous gene in unmodified wild-type cells of the same cell type as the cloaked cell (e.g., stem cells, such as embryonic stem cells from the same species).
  • stem cells such as embryonic stem cells from the same species.
  • all 8 cloaking transgenes are expressed at a level that is greater (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or 100-fold higher or more) than the expression level of the endogenous gene in a wild-type version of the cell (e.g., a stem cell, e.g., an embryonic stem cell from the same species as the cloaked cell).
  • Gene expression can be evaluated through direct comparison to isolated ES cells, or compared to stem cell expression (e.g., ES cell expression) in the Project Grandiose dataset (www.stemformatics.org/project_grandiose). Gene expression can be measured using techniques known in the art (e.g., quantitative polymerase chain reaction (qPCR)).
  • the method comprises providing a cell; and expressing in the cell a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting and whose function is to mitigate function of graft attacking leukocyte and NK cell activation or act as a defense mechanism against attacking leukocytes.
  • the set of transgenes comprises 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).
  • the set of transgenes genes comprises Pd-L1, H2-M3, Cd47, Cd200, FasL, Ccl21 b, Mfge8, and Spi6.
  • the method further comprises expressing one or more of the following transgenes in the cell: TGF- ⁇ , Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFN ⁇ R1 d39.
  • TGF- ⁇ or the biologic is local acting.
  • the cell is a stem cell, a cell amenable to genome editing, and/or a source of therapeutic cell type (e.g., a cell that can be differentiated into a lineage restricted cell for cell therapy, or a cell of a desired target tissue).
  • a source of therapeutic cell type e.g., a cell that can be differentiated into a lineage restricted cell for cell therapy, or a cell of a desired target tissue.
  • the cell is an embryonic stem cell, an induced pluripotent stem cell, an adult stem cell, a tissue-specific stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem or progenitor cells, germline stem cell, a lung stem or progenitor cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, a multipotent stem cell, an amniotic stem cell, a cord blood stem cell, or a neural stem or progenitor cell.
  • the cell is derived from a target tissue, e.g., skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.
  • a target tissue e.g., skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.
  • the cell is a fibroblast, an epithelial cell, or an endothelial cell.
  • the cell may be a vertebrate cell, for example, a mammalian cell, such as a human or mouse cell.
  • the cell that is 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) is a cell in the tissue or organ to be transplanted.
  • the cloaked cells e.g., cloaked stem cells
  • one million to one hundred billion cloaked cells e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10°, 2 ⁇ 10°, 3 ⁇ 10°, 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10°, 7 ⁇ 10 9 , 8 ⁇ 10 9 , 9 ⁇ 10 9 , 1 ⁇ 10 10 , 2 ⁇ 10 10 , 3 ⁇ 10 10 , 4 ⁇ 10 9 , 5 ⁇
  • the cloaked cells described herein can be further modified to express a therapeutic agent.
  • the therapeutic agent is a protein.
  • the therapeutic protein can be a wild type form of a protein that is deficient in a subject, such as a protein that is mutated or produced in insufficient quantity (e.g., produced at low levels or not produced) by the subject's cells.
  • the therapeutic protein is an inhibitory antibody (e.g., an antibody that blocks or neutralizes protein function).
  • the cloaked cells may be modified to produce an inhibitory antibody to treat a subject having or at risk of developing a disease or condition related to overproduction or aberrant production of a protein (e.g., production by cells that do not normally produce the protein, production of a protein at a time or in a location at which the protein is not normally produced, or production of an excessive amount of a protein).
  • the therapeutic antibody is an agonist antibody (e.g., an activating antibody).
  • the agonist antibody can act by binding to and activating an endogenous receptor (e.g., inducing or increasing signaling downstream of receptor activation or changing the conformation of the endogenous receptor to an open or active state).
  • the cloaked cells may be modified to produce an agonist antibody to treat a subject having or at risk of developing a disease or condition related to under activation of a receptor or signaling pathway.
  • the cloaked cells can be modified to produce the therapeutic protein or antibody using the methods described herein or using other methods known by those of skill in the art.
  • Cloaked cells that produce a secreted protein or antibody can be delivered as circulating cells, injected into the tissue, organ, or body site in need of the therapeutic protein or antibody, or injected subcutaneously to produce a cloaked subcutaneous tissue.
  • Cloaked cells that produce a transmembrane or membrane-bound protein can be injected at or near the site of the endogenous cells that respond to the therapeutic protein.
  • the cloaked cells described herein provide a wild-type copy of a gene that is mutated in the subject (e.g., the cloaked cell is a “wild-type cell” that does not have the genetic cause of the disease and that expresses one, two, three, four, five, six, seven or all eight of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6)).
  • Such cells can be used to treat subjects having a disease or condition caused by a mutation in an endogenous gene (e.g., subjects having a metabolic disorder associated with one or more mutations described herein below).
  • a list of exemplary therapeutic agents that can be administered with or produced by cloaked cells and the associated diseases or conditions that can be treated using these therapeutic agents are provided in Table 2 below.
  • Exemplary therapeutic agents that can be administered with or expressed by cloaked cells to treat disease Disease or Condition Therapeutic Agent Diabetes, altered Insulin, insulotropin, glucagon glycemic states Skeletal growth Human growth hormone retardation Anemia Erythropoietin (EPO), hemoglobins Obesity Ob gene translation product (leptin) Immunodeficiency Adenosine deaminase, purine nucleoside (e.g., AIDS) phosphorylase, CD-4 Hemophilia A Factor VIII Hemophilia B Factor IX Emphysema ⁇ 1 -antitrypsin Hypercholesterolemia LDL receptor protein Disease or Condition Therapeutic Agent Pernicious anemia Intrinsic factor Hypoalbuminemia Albumin Gaucher's disease B-glucosidase (glucocerebrosidase) Cystic fibrosis CF transmembrane conductance regulator Cardiovascular Tissue Plasminogen Activator (tPA), disease urokina
  • VEGF- Trap parental e.g., VEGF-Trap ⁇ B1 , VEGF-Trap ⁇ B2 , VEGF-Trap R1R2 , e.g., aflibercept
  • soluble forms of VEGF receptors e.g., soluble VEGFR-1 or NRP-1
  • platelet factor-4 e.g., platelet factor-4
  • prolactin e.g., prolactin
  • SPARC e.g., bevacizumab or ranibizumab.
  • Osteoarthritis or TNF ⁇ inhibitors (adalimumab, etanercept, Rheumatoid Arthritis infliximab, golimumab, certolizumab), interleukin- 6 (IL6) receptor inhibitors (e.g., tocilizumab), IL1 receptor inhibitors (e.g., anakinra), or other agents used to treat RA (e.g., abatacept, rituximab) Inflammatory Bowel TNF ⁇ inhibitors (adalimumab, etanercept, Disease, Crohn's infliximab, golimumab, certolizumab), disease, mesalazine, prednisone, azathioprine, Ulcerative Colitis methotrexate Addison's Disease Aldosterone, cortisol, glucocorticoids, mineralocorticoids, androgens Hurler syndrome Alpha-L iduroni
  • the therapeutic agent can be expressed using a constitutive promoter described herein or known by those of skill in the art (e.g., CAG, CMV, or another constitutive promoter). If the therapeutic agent is needed intermittently (e.g., needed during a period of relapse or flare up that occurs during a disease or condition, but not needed when a subject is asymptomatic), it can be expressed by an inducible promoter, which provides the capability of expressing the therapeutic agent only when it is needed.
  • a constitutive promoter described herein or known by those of skill in the art
  • the therapeutic agent is needed intermittently (e.g., needed during a period of relapse or flare up that occurs during a disease or condition, but not needed when a subject is asymptomatic)
  • an inducible promoter which provides the capability of expressing the therapeutic agent only when it is needed.
  • TNF ⁇ inhibitors is TNF ⁇ inhibitors.
  • TNF ⁇ inhibitors are currently used to treat rheumatoid arthritis, but are only administered intermittently during flare-ups of joint inflammation as constitutive administration of TNF ⁇ can lead to systemic immunosuppression. If cloaked cells are modified to express TNF ⁇ inhibitors under the control of an inducible promoter, cloaked cells can be used to deliver TNF ⁇ intermittently, thus, obviating the need for repeated injections. Other therapeutic agents that have potentially adverse effects if administered continuously can also be expressed intermittently using inducible promoters as described herein. Exemplary inducible expression systems are described below.
  • tetracycline-controlled transcriptional activation based on tetracycline-controlled transcriptional activation.
  • the antibiotic tetracycline or one of its derivatives (e.g., doxycycline) is used to reversibly activate or inhibit gene expression.
  • a tetracycline response element TRE
  • a gene of interest e.g., a therapeutic transgene to be expressed by cloaked cells
  • a protein called rtTA which also needs to be expressed by the cloaked cells, binds to the TRE and activates transcription in the presence of tetracycline or doxycycline.
  • Tet-On Advanced transactivator rtTA2 5 -M2
  • Tet-On 3G Tet-On 3G
  • optogenetics which uses light sensitive proteins to manipulate gene expression.
  • a recent development in optogenetics that can be used to inducibly express therapeutic agents in cloaked cells involves 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 could be used to control the production of therapeutic agents in cloaked cells that are administered subcutaneously, as blue light can be shone onto the skin near the cloaked subcutaneous tissue to induce production of a therapeutic agent by the cloaked cells.
  • a third method of inducibly activating gene expression involves the use of radio waves.
  • the TRPV1 receptor is fused to a GFP binding domain and co-expressed with a form of ferritin that is 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.
  • ferritin interacts with TRPV1 and allows for an influx of calcium, which activates the transcription factor NFAT.
  • Therapeutic agents can be inducibly expressed using this system if they are operably linked to an NFAT-sensitive promoter element, such as SRE-CRE-NFATRE, and co-expressed with TRPV1-GFP and GFP-ferritin.
  • Radio wave-induced expression provides the advantage of being able to induce expression in cells that are further from the outside of the body, as radio waves can pass through tissue.
  • radiogenetics could be used to regulate gene expression in the retina. This method could, therefore, be used to inducibly express therapeutic transgenes in cloaked cells with non-invasive and non-harmful radio waves.
  • Gene expression can also be regulated using destabilization domain systems.
  • a transgene encoding a protein of interest e.g., a therapeutic agent described herein
  • Exemplary destabilizing domains include mutants of the human FK506- and rapamycin-binding protein (FKBP12), which confer instability to the proteins to which they are fused.
  • FKBP12 mutants include N-terminal mutants F15S, V24A, H25R, E60G, and L106P, and C-terminal mutants M66T, R71G, 0100G, 0100N, E102G, and K1051, as characterized in Banaszynski et al., Cell 126:995 (2006), the disclosure of which is incorporated herein by reference as it pertains to FKBP12 destabilizing domains.
  • Destabilizing domains promote protein degradation.
  • a small molecule synthetic ligand can be used to stabilize the destabilizing domain-containing proteins when expression of the protein of interest (e.g., a therapeutic agent) is desired.
  • the small molecule ligand Shield-1 (Shld1) can be used to stabilize FKBP12 mutant-containing proteins by protecting them from degradation.
  • Other destabilizing domains that can be used to regulate expression proteins of interest include mutants of the E. coli dihydrofolate reductase (ecDHFR) and mutants of the human estrogen receptor ligand binding domain (ERLBD), which confer instability resulting in degradation when fused to a protein of interest and can be stabilized by small molecule ligand trimethoprim (TMP), or by CMP8 or 4-hydroxytamoxifen (4OHT), respectively, as described in Iwamoto et al., Chem Biol. 17:981 (2010) and Miyazaki et al., J Am Chem Soc., 134:3942 (2012), the disclosures of each of which are incorporated herein by reference as they pertain to destabilization domain systems.
  • TMP small molecule ligand trimethoprim
  • 4OHT 4-hydroxytamoxifen
  • Another method for inducible activation of gene expression involves the use of the cumate gene-switch system.
  • regulation is mediated by the binding of the repressor (CymR) to the operator site (CuO), placed downstream of a strong constitutive promoter.
  • CaMR repressor
  • CuO operator site
  • Addition of cumate, a small molecule relieves the repression, allowing for expression of the transgene.
  • rcTA reverse-cumate-Trans-Activator
  • a 6-times repeat of a Cumate Operator (6 ⁇ CuO) may be inserted just before the translational start (ATG) of the therapeutic transgene.
  • rcTA cannot bind to the 6 ⁇ CuO, so the transgene encoding the therapeutic agent will not be transcribed because the 6 ⁇ CuO is not active.
  • cumate When cumate is added, it will form a complex with rcTA, which allows for binding to 6 ⁇ CuO and transcription of the transgene encoding the therapeutic agent (Mullick et al., 2006).
  • an inducible gene expression system is the ecdysone inducible system, in which a retinoid X receptor (RXR) and an N-terminal truncation of ecdysone receptor (EcR) fused to the activation domain of Vp16 (VpEcR) are inserted in the 5′ untranslated region of a gene expressed by the cloaked cell such that they are co-expressed by an endogenous promoter.
  • An ecdysone responsive element (EcRE) with a downstream minimal promoter, can be inserted just upstream of the start codon of the transgene encoding the therapeutic agent. Co-expressed RXR and VpEcR can heterodimerize with each other.
  • dimerized RXR/VpEcR cannot bind to EcRE, so the transgene encoding the therapeutic agent is not transcribed.
  • dimerized RXR/VpEcR can bind to EcRE, such that the transgene encoding the therapeutic agent is transcribed (No et al., 1996).
  • ecdysone administration has no apparent effect on mammals, its use for regulating genes should be excellent for transient inducible expression of any gene.
  • the transgene encoding a therapeutic agent can be modified so that it is functionally divided in to parts/domains, such as a 5′ portion and a 3′ portion, and an FKBP peptide sequence can be inserted into each domain.
  • An IRES (internal ribosomal entry site) sequence may be placed between the two domains, which allows for simultaneous transcription of the two different domains to generate two separate proteins. In the absence of a dimerization agent, the two separate domains of the therapeutic agent will be functionally inactive.
  • the FKBP peptides Upon introduction of a dimerization agent, such as rapamycin or AP20187, the FKBP peptides will dimerize, bringing together the 5′ and 3′ domains of the therapeutic agent and reconstituting an active protein (Rollins et al., 2000).
  • a dimerization agent such as rapamycin or AP20187
  • cloaked cells can be modified to produce a VEGF inhibitor, such as VEGF trap (e.g., a soluble decoy receptor described in Holash et al., Proc Natl Acad Sci U.S.A. 99:11383-11398, 2002, incorporated herein by reference, such as aflibercept) to treat age-related macular degeneration (AMD) or retinal dystrophy.
  • VEGF trap is a biologic that binds to and inhibits VEGF, an angiogenic protein that can promote the formation of aberrant blood vessels.
  • VEGF trap is used to treat wet AMD, which features aberrant growth of blood vessels beneath the retina that can lead to retinal detachment and progressive vision loss.
  • VEGF trap is typically delivered by regular injection into the eye.
  • Cloaked cells can be modified to produce VEGF trap or another VEGF inhibitor by expression of a transgene encoding VEGF trap or another VEGF inhibitor operably linked to a constitutive or inducible promoter.
  • Cloaked cells e.g., stem cells
  • a VEGF inhibitor e.g., VEGF trap
  • RPE retinal pigmented epithelium
  • 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).
  • cloaked cells such as dopaminergic neurons or cells (e.g., stem cells) that can be differentiated in vitro to produce dopaminergic neurons using methods known by those of skill in the art, can be administered to subjects suffering from Parkinson's disease, which is characterized by loss of dopaminergic neurons.
  • Parkinson's disease which is characterized by loss of dopaminergic neurons.
  • Twenty five thousand to one hundred thousand cloaked dopaminergic neurons e.g., 25,000, 50,000, 75,000 or 100,000 cloaked dopaminergic neurons
  • Parkinson's disease e.g., stereotactically injected into the substantia nigra
  • the cloaked cells described herein can also be used to treat cardiac infarction (e.g., myocardial infarction, commonly known as a heart attack). Cardiac infarction occurs when blood flow decreases or stops to a part of the heart, causing damage to the heart muscle.
  • cardiac infarction e.g., myocardial infarction, commonly known as a heart attack.
  • Cardiac infarction occurs when blood flow decreases or stops to a part of the heart, causing damage to the heart muscle.
  • cloaked cells e.g., stem cells
  • isolated cardiac muscle cells can be modified to express cloaking transgenes.
  • Five hundred million to five billion cloaked cardiac muscle cells can be administered to a subject by injection into the cardiac muscle to treat a subject who has suffered a cardiac infarction (e.g., to replace dead or damaged cardiac muscle cells).
  • the cloaked cells described herein can be used to treat osteoarthritis or rheumatoid arthritis.
  • Osteoarthritis and rheumatoid arthritis are characterized by joint inflammation, and are commonly treated with anti-inflammatory therapeutics.
  • cloaked cells can be modified to express anti-inflammatory biologics, such as inhibitors of TNF ⁇ (e.g., TNF ⁇ inhibitory antibodies), which are already in clinical use for the treatment of RA.
  • Cloaked cells can be modified to produce an anti-inflammatory biologic, such as a TNF ⁇ inhibitor, by expression of a transgene encoding an anti-inflammatory biologic operably linked to a constitutive or inducible promoter.
  • Cloaked cells e.g., stem cells
  • an anti-inflammatory biologic e.g., a TNF ⁇ inhibitor
  • isolated articular fibroblasts can be modified to express cloaking transgenes and an anti-inflammatory biologic.
  • One million to one hundred million cloaked articular fibroblasts (e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8 cloaked articular fibroblasts) expressing an anti-inflammatory biologic can be injected into an arthritic or inflamed joint (depending on joint size) to treat osteoarthritis or RA.
  • an anti-inflammatory biologic can be injected into an arthritic or inflamed joint (depending on joint size) to treat osteoarthritis or RA.
  • Anti-inflammatory biologics that can be expressed by cloaked cells to treat osteoarthritis or RA include TNF ⁇ inhibitors (adalimumab, etanercept, infliximab, golimumab, certolizumab), interleukin-6 (IL6) receptor inhibitors (e.g., tocilizumab), IL1 receptor inhibitors (e.g., anakinra), or other agents used to treat RA (e.g., abatacept, rituximab).
  • TNF ⁇ inhibitors adalimumab, etanercept, infliximab, golimumab, certolizumab
  • IL6 receptor inhibitors e.g., tocilizumab
  • IL1 receptor inhibitors e.g., anakinra
  • other agents used to treat RA e.g., abatacept, rituximab.
  • the cloaked cells can be used to treat diabetes (e.g., Type 1 or Type 2 diabetes).
  • Type 1 diabetes results from a failure of the pancreas to produce enough insulin.
  • Type 2 diabetes begins with insulin resistance, but a lack of insulin may develop as the disease progresses.
  • cloaked cells can be modified to express insulin, or insulin-expressing cells from a healthy subject (e.g., pancreatic beta cells from a subject without diabetes) can be modified to express one or more (e.g., one, two, three, four, five, six, seven or all eight) of cloaking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject with diabetes.
  • Cloaked cells can be modified to produce insulin by expression of a transgene encoding insulin operably linked to a constitutive or inducible promoter.
  • Cloaked cells e.g., stem cells
  • insulin producing cells e.g., pancreatic beta cells
  • isolated pancreatic beta cells can be modified to express cloaking transgenes and, optionally, to express a transgene encoding insulin.
  • Eight hundred million to three billion cloaked pancreatic beta cells e.g., 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , or 3 ⁇ 10 9 cloaked pancreatic beta cells
  • insulin e.g., expressing insulin endogenously or expressing insulin due to expression of a transgene encoding insulin
  • a subject to create a cloaked subcutaneous tissue that produces insulin for treating diabetes.
  • the cloaked cells described herein can be used to treat hemophilia.
  • Patients with hemophilia do not produce a functional Factor VIII protein, which is a critical blood component needed for blood clotting. These patients can have severe bleeding, and the standard of care involved multiple injections per week of a purified Factor VIII protein.
  • cloaked cells can be modified to express an additional transgene that encodes Factor VIII. Factor VIII would be expressed constitutively in cloaked cells by being operably linked to a constitutive promoter, such as CMV or CAG.
  • Cloaked cells e.g., stem cells
  • Cloaked cells that express Factor VIII can be differentiated into cells that produce blood coagulation factors (e.g., liver sinusoidal cells or endothelial cells) prior to administration using methods known by those of skill in the art or can be administered without differentiation, or isolated Factor VIII-expressing liver sinusoidal cells or endothelial cells from a healthy subject (e.g., a subject without hemophilia) can be modified to express one or more (e.g., one, two, three, four, five, six, seven or all eight) of cloaking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject with hemophilia.
  • a healthy subject e.g., a subject without hemophilia
  • one or more e.g., one, two,
  • Isolated Factor VIII-expressing liver sinusoidal cells or endothelial cells from a healthy subject that are modified to express one or more cloaking transgenes, can be further modified to express a transgene encoding Factor VIII, if desired to ensure that Factor VIII is expressed at high levels.
  • Eight hundred million to three billion cloaked cells e.g., 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , or 3 ⁇ 10 9 cloaked cells
  • expressing Factor VIII e.g., expressing Factor VIII endogenously or expressing Factor VIII due to expression of a transgene encoding Factor VIII
  • the cloaked cells of the invention can also be used to treat inherited metabolic disorders.
  • In most inherited metabolic disorders a single enzyme is not produced by the body or it is produced in a form that is defective.
  • Inherited metabolic disorders include lysosomal storage disorders, such as Hurler syndrome (deficiency in alpha-L iduronidase), Niemann-Pick disease (mutations in SMPD1, NPC1, or NPC2), Tay-Sachs disease (mutation in HEXA), Gaucher's disease (mutation in GBA gene), Fabry disease (deficiency in alpha galactosidase due to mutation in GLA), and Krabbe disease (deficiency in galactosylceramidase due to mutations in GALC); Galactosemia (deficiency in Galactokinase or galactose-1-phosphate uridyltransferase); Maple syrup urine disease (deficiency in enzyme BCKD); Phenylket
  • cloaked cells can be modified to express the wild-type form of the gene that is mutated in the subject or a transgene encoding the enzyme that is missing or deficient in the subject (see Table 2), or cells from a healthy subject (e.g., a subject that does not have a metabolic disorder) that express the wild-type form of the gene that is mutated in the subject or the enzyme that is deficient in the subject can be modified to express one or more (e.g., one, two, three, four, five, six, seven or all eight) of cloaking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject with a metabolic disorder.
  • a healthy subject e.g., a subject that does not have a metabolic disorder
  • cells from a healthy subject e.g., a subject that does
  • the wild-type form of the gene that is mutated in the subject or a transgene encoding the enzyme that is missing or deficient in the subject can be expressed constitutively in cloaked cells by being operably linked to a constitutive promoter, such as CMV or CAG, or can be inducibly expressed using one of the inducible expression systems described herein.
  • a constitutive promoter such as CMV or CAG
  • Cloaked cells that are modified to express the wild-type form of the gene that is mutated in the subject or the enzyme that is missing or deficient in the subject can be differentiated into cells that normally express the gene or enzyme prior to administration using methods known by those of skill in the art or can be administered without differentiation, or isolated cells from a healthy subject that express the wild-type form of the gene or enzyme that is mutated or deficient in the subject can be modified to express one or more (e.g., one, two, three, four, five, six, seven or all eight) of cloaking transgenes PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) and administered to a subject with a metabolic disorder.
  • HLA-G H2-M3
  • the subject can be evaluated using standard methods to identify the mutated gene related to the metabolic disorder, to ensure that the cloaked cells express the corresponding wild-type gene.
  • Eight hundred million to three billion cloaked cells e.g., 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , or 3 ⁇ 10 9 cloaked cells
  • expressing the wild-type form of the gene that is mutated in the subject can be injected subcutaneously to create a cloaked subcutaneous tissue that produces the corresponding wild-type protein.
  • a method of controlling proliferation of cell at a transplant site in an allogeneic host is provided (e.g., to reduce the tumorigenic potential of a cell at the transplant site or to reduce proliferation of a cell that has become tumorigenic at a transplant site).
  • the method comprises: providing a cell genetically modified to comprise at least one mechanism for providing a local immunosuppression at a transplant site when transplanted in an allogeneic host the cell or a population of such cells; genetically modifying in the cell a cell division locus/loci (CDL), the CDL being one or more loci whose transcription product(s) is expressed by dividing cells (e.g., all dividing cells containing one or more of the immunosuppressive transgenes), the genetic modification of the CDL comprising one or more of: a) an ablation link (ALINK) system, the ALINK system comprising a DNA sequence encoding a negative selectable marker that is transcriptionally linked to a DNA sequence encoding the CDL; and b) an inducible exogenous activator of regulation of a CDL (EARC) system, the EARC system comprising an inducible activator-based gene expression system that is operably linked to the CDL; permitting proliferation of the genetically modified cell comprising the ALINK system by maintaining the genetic
  • Cells that have been modified to control cell division using one or more ALINK and/or EARC systems in one or more CDLs may be referred to as “fail-safe cells”.
  • the number of cells that can be grown from a single fail-safe cell (clone volume) before the cell loses activity of all of the systems (e.g., ALINKs or EARCs) that control cell division through genetic mutation determines the fail-safe volume.
  • the fail-safe volume will depend on the number of ALINKs and the number of ALINK-targeted CDLs. The fail-safe property is further described in Table 3.
  • Geno- Fail-safe Relative (x) to Estimated CDL ALINK type volume a human body weight of # # in CDLs (#cells) 3.72 ⁇ 10 13 cells clones 1 1 het 512 0.0000000000137 1 ⁇ g 1 2 hom 16777216 0.000000451 31 mg 2 3 het, hom 1.374E+11 0.004 0.26 kg 2 4 hom, hom 1.13E+15 30 2100 kg
  • a CDL is a locus identified as an “essential gene” as set forth in Wang et al., 2015, which is incorporated herein by reference as if set forth in its entirety.
  • Essential genes in Wang et al., 2015 were identified by computing a score (i.e., a CRISPR score) for each gene that reflects the fitness cost imposed by inactivation of the gene.
  • a CDL has a CRISPR score (CS) of less than about ⁇ 1.0 (Table 5, column 5).
  • a CDL is a locus/loci that encodes a gene product that is relevant to cell division and/or replication (Table 5, column 6).
  • a CDL is a locus/loci that encodes a gene product that is relevant to one or more of: i) cell cycle; ii) DNA replication; iii) RNA transcription and/or protein translation; and iv) metabolism (Table 5, column 7).
  • a CDL is one or more cyclin-dependent kinases that are involved with regulating progression of the cell cycle (e.g., control of G1/S G2/M and metaphase-to-anaphase transition), such as CDK1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9 and/or CDK11 (Morgan, 2007).
  • a CDL is one or more cyclins that are involved with controlling progression of the cell cycle by activating one or more CDK, such as, for example, cyclinB, cyclinE, cyclinA, cyclinC, cyclinD, cyclinH, cyclinC, cyclinT, cyclinL and/or cyclinF (FUNG and POON, 2005).
  • a CDL is one or more loci involved in the anaphase-promoting complex that controls the progression of metaphase to anaphase transition in the M phase of the cell cycle (Peters, 2002).
  • a CDL is one or more loci involved with kinetochore components that control the progression of metaphase to anaphase transition in the M phase of the cell cycle (Fukagawa, 2007). In an embodiment, a CDL is one or more loci involved with microtubule components that control microtubule dynamics required for the cell cycle (Cassimeris, 1999).
  • a CDL is a locus/loci involved with housekeeping.
  • the term “housekeeping gene” or “housekeeping locus” refers to one or more genes that are required for the maintenance of basic cellular function. Housekeeping genes are expressed in all cells of an organism under normal and patho-physiological conditions.
  • a CDL is a locus/loci that encodes a gene product that is relevant to cell division and/or proliferation and has a CRISPR score of less than about ⁇ 1.0.
  • a CDL is a locus/loci that encodes a gene product that is relevant to one or more of: i) cell cycle; ii) DNA replication; iii) RNA transcription and/or protein translation; and iv) metabolism, and has a CRISPR score of less than about ⁇ 1.0.
  • the CDL may also be a housekeeping gene.
  • 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 CDLs are Cdk1/CDK1 and Top2A/TOP2A or Cdk1/CDK1 and Eef2/EEF2.
  • a cell can be modified to be a “fail-safe” cell by linking the expression of a CDL with that of a DNA sequence encoding a negative selectable marker, thereby allowing drug-induced ablation of mitotically active cells expressing both the CDL and the negative selectable marker.
  • Ablation of proliferating cells may be desirable, for example, when cell proliferation is uncontrolled and/or accelerated relative to a cell's normal division rate (e.g., uncontrolled cell division exhibited by cancerous cells), or when therapeutic need for the cells has passed.
  • Ablation of proliferating cells may be achieved via a genetic modification to the cell, referred to herein as an “ablation link” (ALINK), which links the expression of a DNA sequence encoding a negative selectable marker to that of a CDL, thereby allowing elimination or sufficient inhibition of ALINK-modified proliferating cells consequently expressing the CDL locus (sufficient inhibition being inhibition of cell expansion rate to a rate that is too low to contribute to tumour formation).
  • ALINK ablation link
  • Cells may be modified to comprise homozygous, heterozygous, hemizygous or compound heterozygous ALINKS.
  • a negative selectable marker may be introduced into all alleles functional of a CDL.
  • a negative selectable marker may be introduced into all functional alleles of a CDL.
  • the fail-safe system can be used to eliminate all of the cloaked cells, if desired.
  • An ALINK may be inserted in any position of CDL, which allows co-expression of the CDL and the negative selectable marker.
  • the ALINK system comprises a herpes simplex virus-thymidine kinase/ganciclovir system, a cytosine deaminase/5-fluorocytosine system, a carboxyl esterase/irinotecan system or an iCasp9/AP1903 system.
  • DNA encoding a negatively selectable marker may be inserted into a CDL (e.g., CDK1) in a host cell, such that expression of the negative selectable marker causes host cells expressing the negative selectable marker and, necessarily, the CDL, to be killed in the presence of an inducer (e.g., prodrug) of the negative selectable marker (e.g., ganciclovir (GCV)).
  • an inducer e.g., prodrug
  • GCV ganciclovir
  • host cells modified with the ALINK will produce thymidine kinase (TK) and the TK protein will convert GCV into GCV monophosphate, which is then converted into GCV triphosphate by cellular kinases.
  • GCV triphosphate incorporates into the replicating DNA during S phase, which leads to the termination of DNA elongation and cell apoptosis (Halloran and Fenton, 1998).
  • a modified HSV-TK gene (PreuR et al., 2010) is disclosed herein as one example of DNA encoding a negative selectable marker that may be used in an ALINK genetic modification to selectively ablate cells comprising undesirable cell division rate.
  • negative selectable systems could be used in the tools and/or methods provided herein.
  • Various negative selectable marker systems are known in the art (e.g., dCK.DM (Neschadim et al., 2012)).
  • GEPT gene-direct enzyme/prodrug therapy
  • GEPT aims to improve therapeutic efficacy of conventional cancer therapy with no or minimal side-effects
  • GEPT involves the use of viral vectors to deliver a gene into cancer cells or into the vicinity of cancer cells in an area of the cancer cells that is not found in mammalian cells and that produces enzymes, which can convert a relatively non-toxic prodrug into a toxic agent.
  • HSV-TK/GCV cytosine deaminase/5-fluorocytosine
  • CE/CPT-11 carboxyl esterase/irinotecan
  • TK/GCV Herpes Simplex Virus type 1 thymidine kinase/ganciclovir
  • the CD/5-FC negative selectable marker system is a widely used “suicide gene” system.
  • Cytosine deaminase (CD) is a non-mammalian enzyme that may be obtained from bacteria or yeast (e.g., from Escherichia coli or Saccharomyces cerevisiae , respectively) (Ramnaraine et al., 2003).
  • CD catalyzes conversion of cytosine into uracil and is an important member of the pyrimidine salvage pathway in prokaryotes and fungi, but it does not exist in mammalian cells.
  • 5-fluorocytosine (5-FC) is an antifungal prodrug that causes a low level of cytotoxicity in humans (Denny, 2003).
  • CD catalyzes conversion of 5-FC into the genotoxic agent 5-FU, which has a high level of toxicity in humans (Ireton et al., 2002).
  • the CE/CPT-11 system is based on the carboxyl esterase enzyme, which is a serine esterase found in a different tissues of mammalian species (Humerickhouse et al., 2000).
  • the anti-cancer agent CPT-11 is a prodrug that is activated by CE to generate an active referred to as 7-ethyl-10-hydroxycamptothecin (SN-38), which is a strong mammalian topoisomerase I inhibitor (Wierdl et al., 2001).
  • SN-38 induces accumulation of double-strand DNA breaks in dividing cells (Kojima et al., 1998).
  • a negative selectable marker system is the iCasp9/AP1903 suicide system, which is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) to allow chemical dimerization using a small molecule AP1903, which has tested safely in humans.
  • FKBP human FK506 binding protein
  • Administration of the dimerizing drug induces apoptosis of cells expressing the engineered caspase 9 components.
  • This system has several advantages, such as, for example, including low potential immunogenicity, since it consists of human gene products, the dimerizer drug only effects the cells expressing the engineered caspase 9 components (Straathof et al., 2005).
  • the iCasp/AP1903 suicide system is being tested in clinical settings (Di Stasi et al., 2011).
  • the negative selectable marker system of the ALINK system could be replaced with a proliferation antagonist system.
  • proliferation antagonist refers to a natural or engineered compound(s) whose presence inhibits (completely or partially) division of a cell.
  • Omomyc ER is the fusion protein of MYC dominant negative Omomyc with mutant murine estrogen receptor (ER) domain.
  • the fusion protein Omomyc ER 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, sequestering them in complexes that are unable to bind the Myc DNA binding consensus sequences (Soucek et al., 2002). As a consequence of the lack of Myc activity, cells are unable to divide (Oricchio et al., 2014).
  • TAM tamoxifen
  • A-Fos a dominant negative to activation protein-1 (AP1) (a heterodimer of the oncogenes Fos and Jun) that inhibits DNA binding in an equimolar competition (Olive et al., 1997).
  • A-Fos can also be fused to ER domain, rendering its nuclear localization to be induced by TAM.
  • Omomyc ER /tamoxifen or A-Fos ER /tamoxifen could be a replacement for TK/GCV to be an ALINK.
  • a cell can also be modified to be “fail-safe” by operably linking the CDL with an EARC, such as an inducible activator-based gene expression system.
  • EARC such as an inducible activator-based gene expression system.
  • the CDL will only be expressed (and the cell can only divide) in the presence of the inducer of the inducible activator-based gene expression system.
  • EARC-modified cells stop dividing, significantly slow down, or die in the absence of the inducer, depending on the mechanism of action of the inducible activator-based gene expression system and CDL function.
  • Cells may be modified to comprise homozygous or compound heterozygous EARCs or may be altered such that only EARC-modified alleles can produce functional CDLs.
  • an EARC modification may be introduced into all alleles of a CDL, for example, to provide a mechanism for cell division control.
  • An EARC may be inserted in any position of CDL that permits co-expression of the CDL and the activator component of the inducible system in the presence of the inducer.
  • an “activator” based gene expression system is preferable to a “repressor” based gene expression system.
  • a repressor if a repressor is used to suppress a CDL a loss of function mutation of the repressor could release CDL expression, thereby allowing cell proliferation. In a case of an activation-based suppression of cell division, the loss of activator function (mutation) would shut down CDL expression, thereby disallowing cell proliferation.
  • the EARC system is a dox-bridge system, a cumate switch inducible system, an ecdysone inducible system, a radio wave inducible system, or a ligand-reversible dimerization system.
  • a dox-bridge may be inserted into a CDL (e.g., CDK1) in a host cell, such that in the presence of an inducer (e.g., doxycycline or “DOX”) the dox-bridge permits CDL expression, thereby allowing cell division and proliferation.
  • CDL e.g., CDK1
  • an inducer e.g., doxycycline or “DOX”
  • Host cells modified with a dox-bridge EARC may comprise a reverse tetracycline Trans-Activator (rtTA) gene (Urlinger et al., 2000) under the transcriptional control of a promoter, which is active in dividing cells (e.g., in the CDL). This targeted insertion makes the CDL promoter no longer available for CDL transcription.
  • rtTA reverse tetracycline Trans-Activator
  • a tetracycline responder element promoter for example TRE (Agha-Mohammadi et al., 2004)
  • TRE Alpha-Mohammadi et al., 2004
  • dox-bridge refers to a mechanism for separating activity of a promoter from a target transcribed region by expressing rtTA (Gossen et al., 1995) by the endogenous or exogenous promoter and rendering the transcription of target region under the control of TRE.
  • rITA refers to the reverse tetracycline transactivator elements of the tetracycline inducible system (Gossen et al., 1995)
  • TRE refers to a promoter consisting of TetO operator sequences upstream of a minimal promoter. Upon binding of rITA to the TRE promoter in the presence of doxycycline, transcription of loci downstream of the TRE promoter increases.
  • the rITA sequence may be inserted in the same transcriptional unit as the CDL or in a different location of the genome, so long as the transcriptional expression's permissive or non-permissive status of the target region is controlled by doxycycline.
  • a dox-bridge is an example of an EARC.
  • inducible activator-based gene expression systems could be used in the tools and or methods provided herein to produce EARC modifications.
  • Various inducible activator-based gene expression systems are known in the art.
  • destabilizing protein domains (Banaszynski et al., 2006) fused with an acting protein product of a coding CDL could be used in conjunction with a small molecule synthetic ligand to stabilize a CDL fusion protein when cell division and/or proliferation is desirable.
  • destabilized-CDL-protein will be degraded by the cell, which in turn would stop proliferation.
  • the stabilizer compound When the stabilizer compound is added, it would bind to the destabilized-CDL-protein, which would not be degraded, thereby allowing the cell to proliferate.
  • transcription activator-like effector (TALE) technology could be combined with dimerizer-regulated expression induction (Pollock and Clackson, 2002).
  • TALE transcription activator-like effector
  • the TALE technology could be used to generate a DNA binding domain designed to be specific to a sequence, placed together with a minimal promoter replacing the promoter of a CDL.
  • the TALE DNA binding domain also extended with a drug dimerizing domain. The latter can bind to another engineered protein having corresponding dimerizing domain and a transcriptional activation domain.
  • a reverse-cumate-Trans-Activator may be inserted in the 5′ untranslated region of the CDL, such that it will be expressed by the endogenous CDL promoter.
  • a 6-times repeat of a Cumate Operator (6 ⁇ CuO) may be inserted just before the translational start (ATG) of CDL.
  • rcTA cannot bind to the 6 ⁇ CuO, so the CDL will not be transcribed because the 6 ⁇ CuO is not active.
  • cumate is added, it will form a complex with rcTA, enabling binding to 6 ⁇ CuO and enabling CDL transcription (Mullick et al., 2006).
  • a retinoid X receptor (RXR) and an N-terminal truncation of ecdysone receptor (EcR) fused to the activation domain of Vp16 (VpEcR) may be inserted in the 5′ untranslated region of a CDL such that they are co-expressed by an endogenous CDL promoter.
  • Ecdysone responsive element (EcRE) with a downstream minimal promoter, may also be inserted in the CDL, just upstream of the starting codon. Co-expressed RXR and VpEcR can heterodimerize with each other.
  • dimerized RXR/VpEcR cannot bind to EcRE, so the CDL is not transcribed.
  • dimerized RXR/VpEcR can bind to EcRE, such that the CDL is transcribed (No et al., 1996).
  • a transient receptor potential vanilloid-1 (TRPV1), together with ferritin, may be inserted in the 5′ untranslated region of a CDL and co-expressed by an endogenous CDL promoter.
  • a promoter inducible by NFAT (NFATre) may also be inserted in the CDL, just upstream of the starting codon. In a normal environment, the NFAT promoter is not active.
  • TRPV1 and ferritin create a wave of Ca ++ entering the cell, which in turn converts cytoplasmatic-NFAT (NFATc) to nuclear-NFAT (NFATn), that ultimately will activate the NFATre and transcribe the CDL (Stanley et al., 2015).
  • a CDL may be functionally divided in to parts/domains: 5′-CDL and 3′CDL, and a FKBP peptide sequence may be inserted into each domain.
  • An IRES (internal ribosomal entry site) sequence may be placed between the two domains, which will be transcribed simultaneously by a CDL promoter but will generate two separate proteins. Without the presence of an inducer, the two separate CDL domains will be functionally inactive.
  • a dimerization agent such as rapamycin or AP20187
  • the FKBP peptides Upon introduction of a dimerization agent, such as rapamycin or AP20187, the FKBP peptides will dimerize, bringing together the 5′ and 3′ CDL parts and reconstituting an active protein (Rollins et al., 2000).
  • the genetically modified cell comprises: a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting and whose function is to mitigate function of graft attacking leukocyte and NK cell activation or act as a defense mechanism against attacking leukocytes.
  • Methods for genetically modifying cell to comprise at least one mechanism for providing a local immunosuppression at a transplant site when transplanted in an allogeneic host the cell or a population of such cells are described, for example, in WO 2016/141480, the entire teachings of which are incorporated herein by reference.
  • the set of transgenes comprises one or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).
  • the set of transgenes genes comprises PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6).
  • the method further comprises expressing one or more of the following transgenes in the cell: TGF- ⁇ , Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFN ⁇ R1 d39.
  • TGF- ⁇ or the biologic is local acting in the graft environment.
  • the cell is a stem cell, a cell amenable to genome editing, or a cell that can serve as a source of a therapeutic cell type (e.g., a cell that can be directed to differentiate into a lineage restricted or terminally differentiated cell that can be used for cell therapy, or a cell of a desired target tissue).
  • a therapeutic cell type e.g., a cell that can be directed to differentiate into a lineage restricted or terminally differentiated cell that can be used for cell therapy, or a cell of a desired target tissue.
  • the cell is an embryonic stem cell, an induced pluripotent stem cell, an adult stem cell, a tissue-specific stem cell, a hematopoietic stem cell, a mesenchymal stem cell, an endothelial stem cell, an epithelial stem cell, an adipose stem or progenitor cells, germline stem cell, a lung stem or progenitor cell, a mammary stem cell, an olfactory adult stem cell, a hair follicle stem cell, a multipotent stem cell, an amniotic stem cell, a cord blood stem cell, or a neural stem or progenitor cell.
  • the cell is derived from a target tissue, e.g., skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.
  • a target tissue e.g., skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach.
  • the cell is a fibroblast, an epithelial cell, or an endothelial cell.
  • the cell may be a vertebrate cell, for example a mammalian cell, such as a human or mouse cell.
  • the host has a degenerative disease or a condition that can be treated with cell therapy.
  • diseases or conditions include, but are not limited to: blindness, arthritis (e.g., osteoarthritis or rheumatoid arthritis), ischemia, diabetes (e.g., Type 1 or Type 2 diabetes), multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, a neurological disease, such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and ALS, an enzyme or hormone deficiency, a metabolic disorder (e.g., a lysosomal storage disorder, Galactosemia, Maple syrup urine disease, Phenylketonuria, a glycogen storage disease, a mitochondrial disorder, Friedrich's ataxia, a peroxisomal disorder, a metal metabolism disorder, or an organic academia), an autoimmune disease (e.g., Psoriasis, Systemic Lupus Erythemato
  • a metabolic disorder e.g.,
  • the cloaked cells described herein may be incorporated into a vehicle for administration into a patient, such as a human patient receiving a transplant or suffering from a disease or condition described herein.
  • Pharmaceutical compositions containing cloaked cells can be prepared using methods known in the art.
  • such compositions can be prepared using, e.g., physiologically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013); incorporated herein by reference), and in a desired form, e.g., in the form of aqueous solutions.
  • the cloaked cells described herein can be administered in any physiologically compatible carrier, such as a buffered saline solution.
  • physiologically compatible carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • Other examples include liquid media, for example, Dulbeccos 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.
  • DMEM Dulbeccos modified eagle's medium
  • sterile saline sterile phosphate buffered saline
  • Leibovitz's medium L15, Invitrogen, Carlsbad, Calif.
  • dextrose in
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the solution is preferably sterile and fluid to the extent that easy syringability exists.
  • the solution is stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi through the use of, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosol, and the like.
  • Solutions of the invention can be prepared by using a pharmaceutically acceptable carrier or diluent and, as required, other ingredients enumerated above, followed by filtered sterilization, and then incorporating the cloaked cells as described herein.
  • a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.
  • compositions comprising cloaked cells in a semi-solid or solid carrier are typically formulated for surgical implantation at the site of transplantation or at the affected site of a disease or condition in the subject. It will be appreciated that liquid compositions also may be administered by surgical procedures.
  • semi-solid or solid pharmaceutical compositions may comprise semi-permeable gels, matrices, cellular scaffolds and the like, which may be non-biodegradable or biodegradable.
  • degradable materials include biocompatible polymers, such as poly(lactic acid), poly(lactic acid-co-glycolic acid), methylcellulose, hyaluronic acid, collagen, and the like.
  • one or more hydrogels are used for the pharmaceutical compositions.
  • the one or more hydrogels may include collagen, atelocollagen, fibrin constructs, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, and poly(ethylene oxide).
  • the hydrogel may be formed of poly(2-hydroxyethyl methacrylate), poly(acrylic acid), self-assembling peptides (e.g., RAD16), poly(methacrylic acid), poly(N-vinyl-2-pyrrolidinone), poly(vinyl alcohol) and their copolymers with each other and with hydrophobic monomers such as methyl methacrylate, vinyl acetate, and the like.
  • hydrophilic polyurethanes containing large poly(ethylene oxide) blocks are also preferred.
  • Other preferred materials include hydrogels comprising interpenetrating networks of polymers, which may be formed by addition or by condensation polymerization, the components of which may comprise hydrophilic and hydrophobic monomers such as those just enumerated.
  • In situ-forming degradable networks are also suitable for use in the invention (see, e.g., Anseth, K S et al. J. Controlled Release, 2002; 78:199-209; Wang, D. et al., Biomaterials, 2003; 24:3969-3980; U.S. Patent Publication 2002/0022676).
  • in situ forming materials are formulated as fluids suitable for injection; then may be induced to form a hydrogel by a variety of means such as change in temperature, pH, and exposure to light in situ or in vivo.
  • the construct contains fibrin glue containing gels.
  • the construct contains atelocollagen containing gels.
  • a polymer used to form a matrix may be in the form of a hydrogel.
  • hydrogels are cross-linked polymeric materials that can absorb more than 20% of their weight in water while maintaining a distinct three-dimensional structure. This definition includes dry cross-linked polymers that will swell in aqueous environments, as well as water-swollen materials.
  • a host of hydrophilic polymers can be cross-linked to produce hydrogels, whether the polymer is of biological origin, semi-synthetic or wholly synthetic.
  • the hydrogel may be produced from a synthetic polymeric material.
  • Such synthetic polymers can be tailored to a range of properties and predictable lot-to-lot uniformity, and represent a reliable source of material that generally is free from concerns of immunogenicity.
  • the matrices may include hydrogels formed from self assembling peptides, such as those discussed in U.S. Pat. Nos. 5,670,483 and 5,955,343, U.S. Patent Application No. 2002/0160471, and PCT Application No. WO 02/062969.
  • hydrogels Properties that make hydrogels valuable in drug delivery applications include the equilibrium swelling degree, sorption kinetics, solute permeability, and their in vivo performance characteristics. Permeability to compounds depends, in part, upon the swelling degree or water content and the rate of biodegradation. Since the mechanical strength of a gel may decline in proportion to the swelling degree, it is also well within the contemplation of the present invention that the hydrogel can be attached to a substrate so that the composite system enhances mechanical strength. In some embodiments, the hydrogel can be impregnated within a porous substrate, so as to gain the mechanical strength of the substrate, along with the useful delivery properties of the hydrogel.
  • the pharmaceutical composition comprises a biocompatible matrix made of natural, modified natural or synthetic biodegradable polymers, including homopolymers, copolymers and block polymers, as well as combinations thereof.
  • biodegradable polymers or polymer classes include any biodegradable polymers discussed within this disclosure, including but not limited to, fibrin, collagen types I, II, III, IV and V, elastin, gelatin, vitronectin, fibronectin, laminin, thrombin, poly(aminoacid), oxidized cellulose, tropoelastin, silk, ribonucleic acids, deoxyribonucleic acids; proteins, polynucleotides, gum arabic, reconstituted basement membrane matrices, starches, dextrans, alginates, hyaluron, chitin, chitosan, agarose, polysaccharides, hyaluronic acid, poly(lactic acid), poly(glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, their derivatives and mixtures thereof.
  • Suitable polymers also include poly(lactide) (PLA) which can be formed of L(+) and D( ⁇ ) polymers, polyhydroxybutyrate, polyurethanes, polyphoshazenes, poly(ethylene glycol)-poly(lactide-co-glycolide) co-polymer, degradable polycyanoacrylates and degradable polyurethanes.
  • PLA poly(lactide)
  • L(+) and D( ⁇ ) polymers polyhydroxybutyrate
  • polyurethanes polyphoshazenes
  • poly(ethylene glycol)-poly(lactide-co-glycolide) co-polymer poly(ethylene glycol)-poly(lactide-co-glycolide) co-polymer
  • degradable polycyanoacrylates degradable polyurethanes.
  • biodegradable polymers or polymer classes include, without limitation, aliphatic polyesters, poly(alkylene oxalates), tyrosine derived polycarbonates, polyiminocarbonates, polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(propylene fumarate), polyfumarates, polydioxanones, polycarbonates, polyoxalates, poly(alpha-hydroxyacids), poly(esters), polyurethane, poly(ester urethane), poly(ether urethane), polyanhydrides, polyacetates, polycaprolactones, poly(orthoesters), polyamino acids, polyamides and blends and copolymers thereof.
  • Additional useful biodegradable polymers include, without limitation stereopolymers of L- and D-lactic acid, copolymers of bis(para-carboxyphenoxy)propane and sebacic acid, sebacic acid copolymers, copolymers of caprolactone, poly(lactic acid)/poly(glycolic acid)/polyethyleneglycol copolymers, copolymers of polyurethane and poly(lactic acid), copolymers of alpha-amino acids, copolymers of alpha-amino acids and caproic acid, copolymers of alpha-benzyl glutamate and polyethylene glycol, copolymers of succinate and poly(glycols), polyphosphazene, poly(hydroxyalkanoates) and mixtures thereof. Binary and ternary systems also are contemplated.
  • the material used to form a matrix is desirably configured so that it: (1) has mechanical properties that are suitable for the intended application; (2) remains sufficiently intact until tissue has in-grown and healed; (3) does not invoke an inflammatory or toxic response; (4) is metabolized in the body after fulfilling its purpose; (5) is easily processed into the desired final product to be formed; (6) demonstrates acceptable shelf-life; and (7) is easily sterilized.
  • the population of cloaked cells can be administered by use of a scaffold.
  • the composition, shape, and porosity of the scaffold may be any described above.
  • these three-dimensional biomaterials contain the living cells attached to the scaffold, dispersed within the scaffold or incorporated in an extracellular matrix entrapped in the scaffold. Once implanted into the target region of the body, these implants become integrated with the host tissue, wherein the transplanted cells gradually become established.
  • Non-limiting examples of scaffolds that may be used include textile structures, such as weaves, knits, braids, meshes, non-wovens, and warped knits; porous foams, semi-porous foams, perforated films or sheets, microparticles, beads, and spheres and composite structures being a combination of the above structures.
  • Nonwoven mats may, for example, be formed using fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (PGA/PLA), sold under the tradename VICRYL sutures (Ethicon, Inc., Somerville, N.J.).
  • Foams composed of, for example, poly(epsilon-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer, formed by processes such as freeze-drying, or lyophilized, as discussed in U.S. Pat. No. 6,355,699, also may be utilized.
  • the framework is a felt, which can be composed of a multifilament yarn made from a bioabsorbable material.
  • the yarn can be made into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling.
  • cells are seeded onto foam scaffolds that may be used as composite structures.
  • the framework may be molded into a useful shape, such as to fill a tissue void.
  • the framework can therefore be shaped to not only provide a channel for neural growth, but also provide a scaffold for the supporting and surrounding tissues, such as vascular tissue, muscle tissue, and the like.
  • the population of cells may be cultured on pre-formed, non-degradable surgical or implantable devices.
  • compositions may include preparations made from cloaked cells that are formulated with a pharmaceutically acceptable carrier or medium.
  • suitable pharmaceutically acceptable carriers include any discussed within this disclosure, including but not limited to, water, salt solution (such as Ringers solution), alcohols, oils, gelatins, polyvinyl pyrrolidine, carbohydrates such as lactose, amylose, or starch, fatty acid esters, and hydroxymethylcellulose.
  • Such preparations can be sterilized, and if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring agents.
  • Pharmaceutical carriers suitable for use in the present invention are known in the art and are described, for example, in Pharmaceutical Sciences (17 th Ed., Mack Pub. Co., Easton, Pa.) and WO 96/05309.
  • the cloaked 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 transplant, or a subject having a disease or condition described herein) by a variety of routes, such as local administration to or near the site of a transplant, local administration to the site affected by the disease or condition (e.g., injection to a joint for treating RA, injection into the subretinal space for treating wet AMD, direct administration to the central nervous system (CNS) (e.g., intracerebral, intraventricular, intrathecal, intracisternal, or stereotactic administration) for treating a neurological disease, such as Parkinson's disease, direct injection into the cardiac muscle for treating cardiac infarction), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and oral administration.
  • routes such
  • compositions may be administered once, or more than once (e.g., once annually, twice annually, three times annually, bi-monthly, or monthly).
  • the cloaked cells may be administered by any means that places the population of cells in a desired location, including catheter, syringe, shunt, stent, microcatheter, pump, implantation with a device, or implantation with a scaffold.
  • the population of cells can be incubated in the presence of one or more factors, or under conditions, that stimulate stem cell differentiation into a desired cell type (e.g., a neuron, a cardiac muscle cell, an RPE cell, an insulin producing cell, a blood coagulation factor producing cell, an articular fibroblast, or other cell types described herein).
  • a desired cell type e.g., a neuron, a cardiac muscle cell, an RPE cell, an insulin producing cell, a blood coagulation factor producing cell, an articular fibroblast, or other cell types described herein.
  • Such factors include growth or trophic factors, chemokines, cytokines, cellular products, demethylating agents, and other stimuli which are known to stimulate differentiation, for example, of stem cells along angiogenic, hemangiogenic, vasculogenic, skeletal muscle, vascular smooth muscle, pericyte, neuronal, or vascular endothelial pathways or lineages.
  • the composition administered to the patient includes a population of cloaked cells with one or more factors that stimulate cell differentiation into a desired cell type, where the cell differentiation occurs in vivo at the tissue site.
  • the cloaked cells can be differentiated into an organ or tissue in vitro using methods known by those of skill in the art and administered to a subject in need of an organ or tissue transplant.
  • cells of a specific cell type are collected from the patient or from a donor (e.g., from an HLA-matched or mis-matched donor that is, e.g., free of the disease or condition), modified to express one or more (e.g., one, two, three, four, five, six, seven, or eight) cloaking transgenes, and subsequently administered to a subject.
  • a donor e.g., from an HLA-matched or mis-matched donor that is, e.g., free of the disease or condition
  • Such an approach is useful for treating subjects carrying a mutation in a particular gene, as the cloaked donor cells can endogenously express the wild-type version of the gene, or for subjects deficient in a particular secreted protein or enzyme (e.g., using cloaked donor cells that endogenously
  • This approach can also be used for treatment of subjects receiving an organ or tissue transplant, as 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) of the cloaking transgenes before the transplant is performed.
  • one or more e.g., one, two, three, four, five, six, seven, or eight
  • Subjects that may be treated as described herein are subjects that have received a transplant, or subjects having a disease or condition described herein (e.g., wet AMD or retinal dystrophy, a neurodegenerative disease, such as Parkinson's disease, cardiac infarction, osteoarthritis or RA, diabetes, hemophilia, a metabolic disorder, or a disease or condition listed in Table 2).
  • the cells, compositions, and methods described herein can be used to treat a disease or condition caused by or associated with loss of cells, a mutation or deficiency in a protein, or aberrant production of a protein, which could be treated using cell replacement protein or cellular therapy, production of a therapeutic protein, production of an agonist antibody, or production of an inhibitory antibody.
  • the methods described herein may include a step of screening a subject for mutations in genes associated with deficient protein production prior to treatment with or administration of the compositions described herein.
  • a subject can be screened for a genetic mutation using standard methods known to those of skill in the art (e.g., genetic testing).
  • the methods described herein may also include a step of evaluating the symptoms of the disease or condition in a subject prior to treatment with or administration of the cloaked cells or compositions described herein. The subject can then be evaluated using the same diagnostic tests after administration of the cloaked cells or compositions to determine whether the subject's condition has improved.
  • the compositions and methods described herein may be administered as a preventative treatment to patients who have received a tissue or organ transplant before the patient shows any signs of tissue or organ rejection.
  • the cloaked cells, compositions, and methods described herein can be used to replace dead or dying cells in a subject (e.g., to replace neurons in a subject suffering from a neurodegenerative disease, or to replace cardiac muscle cells in a subject who has had a myocardial infarction).
  • the cloaked cells, compositions, and methods described herein can also be used to provide immunosuppression in the region of a tissue or organ transplant, or to reduce the risk of rejection of the tissue or organ transplant.
  • Cloaked cells that express a therapeutic agent, such as a protein or agonist antibody, compositions including such cells, or methods of administering such cells may be used to replace or supply wild type versions of proteins that are mutated or deficient in a subject (e.g., proteins that are produced but do not function correctly due to a genetic mutation, such as truncated proteins or proteins with altered charge, polarity, or binding properties; or proteins that are not produced or that are produced in insufficient quantities, e.g., deficient protein production that is associated with a disease or condition in Table 2).
  • proteins that are mutated or deficient in a subject e.g., proteins that are produced but do not function correctly due to a genetic mutation, such as truncated proteins or proteins with altered charge, polarity, or binding properties; or proteins that are not produced or that are produced in insufficient quantities, e.g., deficient protein production that is associated with a disease or condition in Table 2.
  • Cloaked cells that express a therapeutic agent, such as an inhibitory or neutralizing antibody, compositions including such cells, or methods of administering such cells may be used to block or neutralize proteins that are overexpressed in a subject or proteins that are aberrantly produced (e.g., proteins that are produced in at a time or in a location that differs from production of that protein in healthy subjects, e.g., aberrant protein production that is associated with a disease or condition listed in Table 2).
  • proteins that are produced in at a time or in a location that differs from production of that protein in healthy subjects e.g., aberrant protein production that is associated with a disease or condition listed in Table 2.
  • Treatment may include administration of cloaked cells or a composition containing cloaked cells in various unit doses.
  • Each unit dose will ordinarily contain a predetermined-quantity of the cloaked cells described herein.
  • the quantity to be administered, and the particular route of administration and formulation, are within the skill of those in the clinical arts.
  • a unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time. Dosing may be performed using a catheter, syringe, shunt, stent, microcatheter, pump, implantation with a device, or implantation with a scaffold.
  • the number of cells administered may vary depending on whether the cells are administered to a tissue, organ, or body site associated with a disease or injury, or are administered subcutaneously to produce a cloaked subcutaneous tissue.
  • the cloaked cells may be administered to the patient at a dose of, for example 1 ⁇ 10 4 cells to 1 ⁇ 10 10 cells (e.g., 1 ⁇ 10 4 , 2 ⁇ 10 4 , 3 ⁇ 10 4 , 4 ⁇ 10 4 , 5 ⁇ 10 4 , 6 ⁇ 10 4 , 7 ⁇ 10 4 , 8 ⁇ 10 4 , 9 ⁇ 10 4 , 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 6
  • the number of cells administered will depend on the size of the recipient tissue, organ, or body site.
  • 2.5 ⁇ 10 4 to 1 ⁇ 10 6 cells e.g., 2.5 ⁇ 10 4 , 3 ⁇ 10 4 , 4 ⁇ 10 4 , 5 ⁇ 10 4 , 6 ⁇ 10 4 , 7 ⁇ 10 4 , 8 ⁇ 10 4 , 9 ⁇ 10 4 , or 1 ⁇ 10 6 cells
  • 1 ⁇ 10 6 to 1 ⁇ 10 8 cells e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8
  • 8 ⁇ 10 8 cells to 3 ⁇ 10 9 cells can be administered (e.g., injected) subcutaneously.
  • Cloaked cells can be administered in two or more doses (e.g., two, three, four, five, or more different doses, e.g., to joints of different sizes in a patient with RA) or at the same dose two or more times (e.g., two, three, four, five, six, or more times over the course of an hour, day, week, month, or year).
  • the cloaked cells described herein are administered as a tissue (e.g., a tissue that has been grown and/or differentiated in vitro from cloaked cells).
  • the cloaked tissue is administered (e.g., implanted) with a gel, biocompatible matrix, or scaffold.
  • compositions described herein are administered in an amount sufficient to prevent or reduce transplant rejection or to improve symptoms of a disease or condition listed in Table 2 (e.g., to reduce symptoms of osteoarthritis or RA (e.g., reduce inflammation, joint pain, stiffness, or immobility); reduce symptoms of retinal dystrophy or wet AMD (e.g., improve vision, slow or reduce vascularization of the eye); reduce symptoms of Parkinson's disease (e.g., reduce tremors, rigidity, bradykinesia, or improve posture or gait); reduce symptoms of diabetes (e.g., improve insulin levels, reduce the need for regular insulin injections); reduce symptoms of cardiac infarction (e.g., improve heart function, reduce infarct size); reduce symptoms of hemophilia (e.g., increase level of blood coagulation factors, such as Factor VIII, reduce excessive bleeding, reduce bruising, reduce nosebleeds, reduce joint pain or swelling); or reduce symptoms of metabolic disorders (e.g., increase appetite, growth, or weight gain, or reduce
  • Transplant rejection may be evaluated using standard methods known by those of skill in the art and may be reduced by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) compared to rates of transplant rejection typically observed without treatment.
  • administration of the cloaked cells or compositions described herein results in an equivalent outcome in transplant rejection as that observed in subjects administered immunosuppressive agent(s).
  • Symptoms of diseases and conditions described herein can be evaluated using standard methods known to those of skill in the art and may be reduced (e.g., the subject's condition may be improved) by 5% or more (e.g., 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) compared to symptoms prior to administration of the cloaked cells or compositions described herein. These effects may occur, for example, within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, or more, following administration of the compositions described herein.
  • the patient may be evaluated 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more following administration of the cloaked cell or composition depending on the dose and route of administration used for treatment. Depending on the outcome of the evaluation, the patient may receive additional treatments.
  • the cloaked cells described herein are administered in combination with one or more additional therapeutic agents.
  • the additional therapeutic agent(s) can be administered prior to administration of the cloaked cells, after administration of the cloaked cells, or concurrently with administration of the cloaked cells.
  • the cloaked cells and additional therapeutic agents can also be administered simultaneously via co-formulation.
  • the cloaked cells and therapeutic agent(s) can also be administered sequentially, such that the action of the cloaked cells and therapeutic agent(s) overlaps and their combined effect is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with the cloaked cells or therapeutic agent delivered alone or in the absence of the other.
  • the effect of the cloaked cells and therapeutic agent(s) can be partially additive, wholly additive, or greater than additive (e.g., synergistic).
  • Sequential or substantially simultaneous administration of cloaked cells and therapeutic agent(s) can be effected by any appropriate route including, but not limited to oral routes, intravenous routes, intramuscular routes, local routes, or subcutaneous routes.
  • the cloaked cells and therapeutic agent(s) can be administered by the same route or by different routes. For example, cloaked cells may be administered by subcutaneous injection while the additional therapeutic agent is administered orally.
  • the cloaked cells may be administered immediately, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to, 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to hours 16, up to 17 hours, up 18 hours, up to 19 hours up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours up to 24 hours or up to 1-7, 1-14, 1-21 or 1-30 days before or after the additional therapeutic agent.
  • the additional therapeutic agent is an immunosuppressive agent(s) commonly given for organ or tissue transplant.
  • the immunosuppressive agent(s) may be an agent that is given immediately after transplantation to prevent acute rejection (e.g., methylprednisolone, atgam, thymoglobulin, OKT3, basiliximab, or daclizumab) or an immunosuppressive agent(s) used for maintenance (e.g., prednisone, a calcineurin inhibitor (e.g., cyclosporine, tacrolimus), Mycophenolate Mofetil, Azathioprine or Rapamycin).
  • a calcineurin inhibitor e.g., cyclosporine, tacrolimus
  • Mycophenolate Mofetil Azathioprine or Rapamycin
  • immunosuppressive agents given after organ transplantation include corticosteroids (e.g., methylprednisolone, dexamethasone, prednisolone), cytotoxic immunosuppressants (e.g., azathioprine, chlorambucil, cyclophosphamide, mercaptopurine, methotrexate), immunosuppressant antibodies (e.g., antithymocyte globulins, basiliximab, infliximab), sirolimus derivatives (e.g., everolimus, sirolimus), and anti-proliferative agents (e.g., mycophenolate mofetil, mycophenolate sodium, and azathioprine).
  • corticosteroids e.g., methylprednisolone, dexamethasone, prednisolone
  • cytotoxic immunosuppressants e.g., azathioprine, chlorambucil, cyclo
  • the cloaked cell(s) is administered to or near the transplant 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) cloaking transgenes, and the immunosuppressive agent(s) is administered as an additional source of immunosuppression, if needed.
  • one or more e.g., one, two, three, four, five, six, seven, or eight
  • the additional agent may be a disease-modifying anti-rheumatic drug (DMARD), a biologic response modifier (a type of DMARD), a corticosteroid, or a nonsteroidal anti-inflammatory medication (NSAID).
  • DMARD disease-modifying anti-rheumatic drug
  • NSAID nonsteroidal anti-inflammatory medication
  • the additional agent is prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, or a biologic such as tofacitinib, adalimumab, abatacept, anakinra, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab or tocilizumab.
  • a biologic such as tofacitinib, adalimumab, abatacept, anakinra, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab or tocilizumab.
  • the additional agent is 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab (Lemtrada), an aminosalicylate (5-aminoalicylic acid, sulfasalazine, mesalamine, balsalazide, olsalazine), an antibiotic, an anti-histamine, Anti-TNF ⁇ (infliximab, adalimumab, certolizumab pegol, natalizumab), azathioprine, belimumab, beta interferon, a calcineurin inhibitor, certolizumab, a corticosteroids, cromolyn, cyclosporin A, cyclosporine, dimethyl fumarate (tecfidera), etanercept, fingolimod (Gilenya), fumaric acid esters, glatiramer acetate (Copa)
  • the cloaked cell(s) could be administered to replace a tissue or organ damaged by the inflammatory or autoimmune-related disease or condition.
  • the cloaked cell(s) administered could be modified to express a biologic therapeutic agent (e.g., an antibody) directed to treatment of a particular inflammatory or autoimmune-related disease or condition, and the additional agent could be a compound or general anti-inflammatory agent (e.g., an NSAID or corticosteroid).
  • a biologic therapeutic agent e.g., an antibody
  • the additional agent could be a compound or general anti-inflammatory agent (e.g., an NSAID or corticosteroid).
  • the additional agent may be one or more of: prednisone, prednisolone and methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide and azathioprine, tofacitinib, adalimumab, abatacept, anakinra, kineret, certolizumab, etanercept, golimumab, infliximab, rituximab or tocilizumab.
  • the cloaked cell(s) administered could be cartilage or bone producing cells of the joints.
  • the cloaked cell(s) can be modified to produce an anti-TNF ⁇ antibody and can be administered in combination with an anti-inflammatory agent (e.g., a corticosteroid).
  • the additional therapeutic agent may be an additional biologic agent (e.g., bevacuzimab, ranibizumab, or aflibercept), photodynamic therapy, or photocoagulation.
  • the cloaked cell(s) administered could be retinal cells (e.g., RPE cells).
  • the cloaked cell(s) can be modified to produce a VEGF inhibitor and can be administered in combination with photodynamic therapy or photocoagulation.
  • the cloaked cells described herein can be administered with carbidopa-levodopa, a dopamine agonist (e.g., pramipexole, ropinirole, rotigotine, or apomorphine), an MAO-B inhibitor (e.g., selegiline or rasagiline), a catechol-O-methyltransferase inhibitor (e.g., entacapone or tolcapone), anticholinergic (e.g., benztropine or trihexyphenidyl), amantadine, or deep brain stimulation.
  • the cloaked cell(s) administered could be dopaminergic neurons.
  • Additional agents for treating cardiac infarction include anticoagulants (e.g., rivaroxaban, dabigatran, apixaban, heparin, warfarin), anti-platelet agents (e.g., aspirin, clopidogrel, dipyramidole, prasugrel, ticagrelor), angiotensin-converting enzyme inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, Lisinopril, moexipril, perindopril, quinapril, Ramipril, trandolapril), angiotensin II receptor blockers (e.g., candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan), angiotensin receptor neprilysin inhibitors (e.g., sacubitril/valsartan), beta blockers (e
  • the additional agent may be an antiviral compound (e.g., vidarabine, acyclovir, gancyclovir, valgancyclovir, nucleoside-analog reverse transcriptase inhibitor (NRTI) (e.g., AZT (Zidovudine), ddl (Didanosine), ddC (Zalcitabine), d4T (Stavudine), or 3TC (Lamivudine)), non-nucleoside reverse transcriptase inhibitor (NNRTI) (e.g., (nevirapine or delavirdine), protease inhibitor (saquinavir, ritonavir, indinavir, or nelfinavir), ribavirin, or interferon); an antibacterial compound; an antifungal compound; an antiparasitic compound.
  • the cloaked cell(s) administered could be immune cells (e.g., cell that could assist in fighting the infectious
  • the additional agent may be insulin, a sulfonylurea (e.g., chlorpropamide, glipizide, glyburide, glimepiride), a biguanide (e.g., metformin), a meglitinide (e.g., repaglinide, nateglinide), a thiazolidinedione (e.g., rosiglitazone, pioglitazone), a DPP-4 inhibitor (sitagliptin, saxagliptin, linagliptin, alogliptin), an SGLT2 inhibitor (e.g., canagliflozin, dapagliflozin), an alpha-glucosidase inhibitor (e.g., acarbose, miglitol), a bile acid sequestrant (e.g., colesevelam), aspirin, or a dietary regimen.
  • the additional therapeutic agent may be a clotting factor, desmopressin, a clot-preserving medication (e.g., an anti-fibrinolytic, e.g., aprotinin, aminocaproic acid, fibrigongen, or tranexamic acid), a fibrin sealant, or physical therapy.
  • a cloaked cell(s) administered could be liver sinusoidal cells or endothelial cells, which can optionally be modified to express a transgene encoding Factor VIII.
  • the additional therapeutic agent may be a coenzyme (e.g., biotin, hydroxycobalamine, riboflavin, pyridoxine, folate, thiamin, ubichinone, tetrahydrobiopterine), a bone marrow transplant, an organ transplant (e.g., a liver, kidney, or heart transplant), hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium-chain triacylglycerols, miglustat, enzyme supplementation therapy, or dietary restriction (e.g., low protein or phenylalanine-restricted diet for subjects with phenylketonuria),
  • the cloaked cell(s) can be cells that carry a wild-type copy of the gene that is mutated in a subject with a metabolic disorder or cells that endogenously produce the enzyme that is deficient in subject with a metabolic disorder (e.g., a liver cell, kidney cell, heart cell, or
  • the additional agent may be a checkpoint inhibitor, a chemotherapeutic drug, a biologic drug, a non-drug therapy (e.g., radiation therapy, cryotherapy, hyperthermia, or surgical excision or tumor tissue), or an anti-cancer vaccine.
  • a chemotherapeutic drug e.g., a biologic drug
  • a non-drug therapy e.g., radiation therapy, cryotherapy, hyperthermia, or surgical excision or tumor tissue
  • an anti-cancer vaccine e.g., radiation therapy, cryotherapy, hyperthermia, or surgical excision or tumor tissue
  • the cloaked cell(s) could be an immune cell that could help fight the cancer (e.g., a macrophage, natural killer cell, dendritic cell, or T cell).
  • Checkpoint inhibitors can be broken down into at least 4 major categories: i) agents such as antibodies that block an inhibitory pathway directly on T cells or natural killer (NK) cells (e.g., PD-1 targeting antibodies such as nivolumab, pidilizumab/CT-011, and pembrolizumab, antibodies targeting TIM-3, and antibodies targeting LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049, or KIR), ii) agents such as antibodies that activate stimulatory pathways directly on T cells or NK cells (e.g., antibodies targeting OX40, GITR, or 4-1 BB), iii) agents such as antibodies that block a suppressive pathway on immune cells or rely on antibody-dependent cellular cytotoxicity to deplete suppressive populations of immune cells (e.g., CTLA-4 targeting antibodies such as ipilimumab or tremelimumab, antibodies targeting VISTA, and antibodies targeting PD-L2 (e.g., a PDL
  • the inhibitor of checkpoint is an inhibitor (e.g., an inhibitory antibody or small molecule inhibitor) of HVEM, CD160, CHK 1, CHK2, B-7 family ligands, or a combination thereof.
  • an inhibitory antibody or small molecule inhibitor of HVEM, CD160, CHK 1, CHK2, B-7 family ligands, or a combination thereof.
  • Such agents described herein can be designed and produced, e.g., by conventional methods known in the art (e.g., Templeton, Gene and Cell Therapy, 2015; Green and Sambrook, Molecular Cloning, 2012).
  • the inhibitor of checkpoint is an inhibitory antibody (e.g., a monospecific antibody such as a monoclonal antibody).
  • the antibody may be, e.g., humanized or fully human.
  • the inhibitor of checkpoint is a fusion protein, e.g., an Fc-receptor fusion protein.
  • the inhibitor of checkpoint is an agent, such as an antibody, that interacts with a checkpoint protein.
  • the inhibitor of checkpoint is an agent, such as an antibody, that interacts with the ligand of a checkpoint protein.
  • Chemotherapeutic agents include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopyyllotoxins, antibiotics, L-asparaginase, topoisomerase inhibitors, interferons, platinum coordination complexes, anthracenedione substituted urea, methyl hydrazine derivatives, adrenocortical suppressant, adrenocorticosteroides, progestins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasing hormone analog.
  • 5-fluorouracil 5-FU
  • leucovorin LV
  • irenotecan oxaliplatin
  • capecitabine paclitaxel
  • doxetaxel chemotherapeutic agents
  • alkylating agents such as thiotepa and cyclosphosphamide
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines such as benzodopa, carboquone, meturedopa, and uredopa
  • ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine
  • acetogenins especially bullatacin and bullatacinone
  • a camptothecin including the synthetic analogue topotecan
  • bryostatin callystatin
  • CC-1065 including its
  • Anti-cancer biologics include cytokines (e.g., interferon or an interleukin (e.g., IL-2)) used in cancer treatment.
  • the biologic is an anti-angiogenic agent, such as an anti-VEGF agent, e.g., bevacizumab.
  • the biologic is an immunoglobulin-based biologic, e.g., a monoclonal antibody (e.g., a humanized antibody, a fully human antibody, an Fc fusion protein or a functional fragment thereof) that agonizes a target to stimulate an anti-cancer response, or antagonizes an antigen important for cancer.
  • Such agents include Rituximab; Daclizumab; Basiliximab; Palivizumab; Infliximab; Trastuzumab; Gemtuzumab ozogamicin; Alemtuzumab; Ibritumomab tiuxetan; Adalimumab; Omalizumab; Tositumomab-I-131; Efalizumab; Cetuximab; Bevacizumab; Natalizumab; Tocilizumab; Panitumumab; Ranibizumab; Eculizumab; Certolizumab pegol; Golimumab; Canakinumab; Ustekinumab; Ofatumumab; Denosumab; Motavizumab; Raxibacumab; Belimumab; Ipilimumab; Brentuximab Vedotin; Pertuzumab; Ado-trastuzumab e
  • the invention also features a kit containing the cloaked cells described herein (e.g., cloaked cells expressing a set of the cloaking transgenes described herein (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 of PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21 b, Mfge8, and Spi6), optionally further expressing one or more of the following transgenes: TGF- ⁇ , Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1,and IFN ⁇ R1 d39).
  • a set of the cloaking transgenes described herein e.g., 1, 2, 3, 4, 5, 6, 7, or 8 of PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21 b, Mfge8, and Spi6
  • TGF- ⁇ e.g.
  • the cloaked cells are further modified to contain one or more systems for regulating cell division (e.g., an ALINK or EARC system), and/or a therapeutic agent (e.g., a transgene encoding a protein or antibody).
  • the cloaked cells may be provided in a pharmaceutical composition.
  • the kit may further include a syringe for administration of the cloaked cells or pharmaceutical composition and instructions for administering the cloaked cells or pharmaceutical composition for treating a disease or condition described herein.
  • Plasmids containing the cDNA sequences of genes involved in allo-tolerance were obtained as follows:
  • AttB-containing cDNA was recombined into pDONR221 vectors (Thermo Fisher, #1256017) by the BP (recombination between attB and attP sites) reaction to create entry (pENTRY) clones ( FIG. 15( b ) ).
  • the BP reaction entails mixing the attB-flanked transgene cDNA with the pDONR221 plasmid in a 1 mL tube, along with buffers and the BP enzyme provided by Invitrogen, where the BP enzyme recombines the GOI into the docking site of the pDONR221 plasmid.
  • Insertion of the transgene into the pDONR221 plasmid was verified by DNA sequencing (TCAG Sequencing Facility at the Centre for Applied Genomics, Toronto). pENTRY clones that contained the GOI were then recombined into destination vectors via the gateway LR (recombination between attL and attR sites) reaction ( FIG. 15( c ) ).
  • the LR reaction entails mixing the GOI-containing pDONR221 plasmid and the destination vector in a 1 mL tube, along with buffers and the LR enzyme provided by Invitrogen, where the LR enzyme recombines the GOI cassette from the pDONR221 plasmid into the docking site of the destination plasmid.
  • Destination vectors which were used for all transgene constructions, contain a CAGG promoter followed by a Gateway entry site, internal ribosomal entry site (IRES) and either a Puromycin resistance selectable marker or a green fluorescent protein (GFP) reporter. The entire cassette is flanked by transposable PB sites. Following LR recombination, the final destination vectors containing the GOI ( FIG. 15( d ) ) were verified by restriction enzyme digestion.
  • IRS internal ribosomal entry site
  • GFP green fluorescent protein
  • Mouse ES cells derived from the inbred C57BL/6N mouse strain 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 acid (NEAA), Glutamax, Penicillin/Streptomycin, Beta-mercapto-ethanol and leukemia inhibitory factor (LIF) (Behringer et al 2014).
  • FBS fetal bovine serum
  • LIF leukemia inhibitory factor
  • Cells were cultured on a feeder layer of MitomycinC-inactivated Murine Embryonic Fibroblasts (MEFs). Cultures were kept in a standard cell culture incubator at 37° C. and 5% CO2.
  • Transfection was performed using JetPRIME reagent (Polyplus, catalog #14-07) per manufacturer protocol, and was done in three steps: 1) transfection with PD-L1-IRES-GFP destination vector only, 2) transfection with all the other transgenes carrying a Puromycin selectable marker, and 3) transfection with an eLuciferase-IRES-GFP transgene.
  • Step 1 Following transfection with PD-L1-IRES-GFP, cells were plated at low density so that after multiple rounds of proliferation 5-6 days later, individual cell clones—existing as cell aggregates (colonies)—were selected based on the intensity of GFP expression and then expanded as a clonal cell culture. The clone with the highest and most consistent GFP expression was chosen for the next step.
  • Step 2 24 hours after transfection with transgenes containing a Puromycin selection marker, Puromycin was added to the culture media. On the third day, cells were plated at clonal density and Puromycin selection was continued until individual colonies were picked and expanded as clones. A large number of these clones were screened in vivo and the one capable of forming a teratoma in an allogeneic setting was designated “NT2”.
  • 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 chosen for further studies.
  • Quantitative PCR was performed using Sensifast mastermix from Bioline, #Bio-98020, gene specific primers and RNA at a 1:50 dilution. Samples were plated in 384 well plates using the Eppendorf epMotion 5070 robot and the quantitative PCR was performed on BioRad CFX384 Real-Time System C1000 Thermal cycler according to standard protocols. qPCR data was captured by BioRad CFX Manager 3.1 software and expression levels calculated with Microsoft Excel.
  • mice were injected into the peritoneal cavity with 50 mg/kg GCV every 2-3 days for 2-3 weeks. This treatment regimen resulted in an initial brief shrinkage of the tumours, followed by stabilization of tumour size at 400-500 mm 2 after 2-3 weeks of treatment. At the endpoint of the experiment, mice were sacrificed and tumours were dissected. A small portion of tissue was snap-frozen for RNA extraction while the rest was fixed in 4% paraformaldehyde.
  • mice that developed teratomas derived from cells transfected with the eLuciferase transgene were injected with 30 mg/mL VivoGlo Luciferin at 100 uL/25 g body mass (Promega #P104C) 10 min before imaging. Animals were anaesthetized with Isoflurane and placed in an IVIS Lumina II imager (Caliper Life Sciences) driven by Living Image software. Exposure times were set between 5 seconds and 5 minutes depending on signal intensity.
  • Transgenes encoding the genes in Table 1 were cloned into expression vectors and sequence verified both by polymerase chain reaction (PCR), restriction enzyme digestion and sequencing, all using standard methods know in the art.
  • a set of constructs containing transgenes Cd47, Cd200, FasL and H2-M3 (Set 1) were transfected into mouse embryonic stem cells derived from the inventors' C57BL/6 mouse ES line (C2). The presence of the transgenes was verified by PCR and expression of the expressed proteins was documented by immunohistochemistry ( FIGS. 1A-D ).
  • a second set of constructs containing transgenes Ccl21, Mfge8, TGF- ⁇ and Spi6 (Set 2) were transfected into ES cells derived from FVB/N (ES line C2).
  • a modified in vitro Mixed Lymphocyte Reaction (MLR) assay was used to screen for the transgene combination resulting in the most efficient inhibition of T-cell activation.
  • Cell lines transfected with Set 1 and Set 2 cloaking transgenes from Example 1 were used.
  • Donor OT-I splenocytes were labeled with carboxyfluorescein succinimidyl ester CFSE and 60,000 cells were added to each well of the 96-well plate.
  • ES or melanoma cells were mixed 10:1 with ova expressing cells. 10,000 of these were added to each well of splenocytes.
  • IL-2 was added as a general activator and T-cell proliferation was measured by flow cytometry 3 days later ( FIGS. 2A-2E ).
  • Cells were initially gated to include CD8+ cells only and all conditions were set up in 4 replicates.
  • FIG. 2A The negative control (splenocytes only) resulted in a baseline 6.12% proliferation rate ( FIG. 2A ).
  • Wildtype B16 melanoma (+10% ova expressing) cells resulted in distinct acceleration of proliferation to 17.1% ( FIG. 2B ), while cloaked cells reduced this proliferation to 9.51% ( FIG. 2C ).
  • FIG. 2D Similar results were obtained for wildtype ( FIG. 2D ) versus cloaked ES cells ( FIG. 2E ).
  • the effect of these transgenes could be evaluated by the MLR alone as these events act on the maturation and physical migration of host APCs to local lymph nodes where they subsequently activate na ⁇ ve T and B cells.
  • the transgene can be identified.
  • wild-type ESCs derived from C57BL/6 mice are not capable of forming teratomas in FVB/N mice.
  • wild-type ESCs derived from the FVB/N background are not capable of forming teratomas in C57BL/6 hosts.
  • ES cell colonies were dissociated with Trypsin, washed once with DMEM without additives and resuspended in Matrigel HC at a concentration of about 50 million cells per milliliter. Recipient mice were anaesthetized and one hundred microliter injected subcutaneously in each flank area. Developing teratomas were followed for 12 weeks and verified by palpation and measurement of volume with caliper.
  • ESCs were transfected with the same transgenes while also adding a Luciferase transgene that can be detected by imaging. Briefly, ES cells were prepared as described above. The presence of viable cells were repeatedly measured by imaging. The images in FIG. 4 were taken on day 17 post injection.
  • the top panel shows the proliferation of immune cloaked cells in isogenic hosts, while the lower panel shows the proliferation of immune cloaked cells in allogeneic hosts.
  • Non-immune cloaked (wild type) ESCs were transplanted into mice carrying an existing immune cloaked tissue and the mouse was evaluated to determine if it could effectively reject a non-immune cloaked graft ( FIG. 6 ).
  • the same mice were imaged several times over a period of 15 days.
  • the graft was not rejected over time.
  • the left mouse in the right panel of FIG. 6 had a pre-existing immune cloaked graft (arrows).
  • Fail-Safe C57BL/6 ES cell line (as described, for example, in WO/2017/141480) was co-transfected with 5 candidate cloaking transgenes (PD-L1, FasL, Cd47, Cd200 and H2-M3), none of these transgene lines resulted in teratomas in allograft settings.
  • the set of co-transfected genes was expanded by three additional candidate cloaking genes: Spi6, Ccl21 b and Mfge8, 38 clonal lines were generated. One of these lines, NT2, created teratomas in an allogeneic recipient (FVB).
  • NT2 was the highest overexpression of Ccl21b (16,000 ⁇ ), FasL (25,000 ⁇ ), Cd200 (1700 ⁇ ), Cd47 (16 ⁇ ), Mfge8 (34 ⁇ ), Spi6 (600 ⁇ ) and H2-M3 (750 ⁇ ) compared to VVT ES cells.
  • PD-L1 although not the highest level expresser among the clones, the 350 ⁇ expression over ES cells was also a significant increase.
  • GCV ganciclovir
  • the cloaking transgenes are expressed under a very strong synthetic promoter, CAG (depicted in the schematic in FIG. 19 ).
  • the CAG promoter is a combination of the cytomegalovirus early enhancer element, the splicer acceptor of the rabbit beta-globin gene, and also the promoter, first exon and first intron of the chicken beta-actin gene.
  • CAG promoter is a combination of the cytomegalovirus early enhancer element, the splicer acceptor of the rabbit beta-globin gene, and also the promoter, first exon and first intron of the chicken beta-actin gene.
  • transcript expression level of the immunomodulatory genes relevant to the cloaking technology varied between ES cell clones. Concentric circles are depicted on a log 10 scale. The thick black line is 1 ⁇ , the next outer ring is 10 ⁇ , and then 100 ⁇ . The innermost ring is 0.1 ⁇ . All values are normalized to positive controls, which were activated leukocytes isolated from murine lymph organs that naturally express the immunomodulatory transgenes. The upper left panel shows wild-type ES cells with no transgenic modifications for reference—they express little or none of the relevant immunomodulatory transgenes. By contrast, clone NT2 and clone 15 (indicated by red squares), both with high expression of the genes, survived in allogenic hosts. All other clones shown in FIG. 9 did not survive in allogenic hosts.
  • the high expression of the cloaking transgenes is also depicted in FIG. 10 .
  • all 8 cloaking transgenes in the NT2 cell line and NT2-derived teratoma had an expression level that was among the top 5% of all genes in the ES cell genome, with 5 of the cloaking transgenes having an expression level in the top 1% of all genes in the ES cell genome.
  • the expression of these genes is much lower in VVT ES cells, as only one of the genes has an expression level among the top 5% of all genes in the genome.
  • FIGS. 13A-13B isogenic host neuronal, bone and columnar epithelium in upper panels ( FIG. 13A ); and allogenic host neuronal, bone, columnar epithelium and blood vessels in lower panels ( FIG. 13A ).
  • FIGS. 14A-140 we tested if the cloaked ES cell were truly pluripotent by testing whether they could form cells from all three germ layers—endoderm, ectoderm, and mesoderm. This was assayed by injecting between 10 6 and 10 7 cloaked ES cells subcutaneously into a mouse and allowing them to proliferate and differentiate into a tissue mass named a teratoma. The teratoma was then removed 3-4 weeks after ES cell injection, and tissue sections cut and stained with H&E. These sections were analyzed under the microscope for cell morphology to determine if all three germ layers were present.
  • FIG. 14D shows a blood vessel, which verifies that these tissues are well-vascularized.
  • Example 10 ES Cells that Express Cloaking Transgenes Produce the Proteins Encoded by the Transgenes
  • FIGS. 16A-16H These data confirm that the proteins encoded by the transgenes are expressed in ES cells at easily detectable levels, which is expected based on the high levels of mRNA expression.
  • Example 11 ES Cells that Express High Levels of Cloaking Transgenes have Typical Morphology and Express Common ES Cell Markers
  • Cloaked ES cells have the typical morphology observed with healthy and pluripotent ES cells ( FIG. 17A ) and also stain positively for alkaline phosphatase ( FIG. 17B ), which is characteristic of healthy and pluripotent ES cells. Furthermore, our cloaked ES cells stained positively for the transcription factor Oct4 ( FIG. 18A ) as well as SSEA ( FIG. 18B ) using fluorescent antibodies, both common markers of normal pluripotent ES cells.
  • ES cells that express high levels of the 8 immunomodulatory cloaking transgenes appear as normal ES cells with respect to their morphology and expression of common ES cell markers.
  • the insets show that staining for Oct4 and SSEA1 (lower left inset) colocalizes with ES cells (visualized using DAPI in upper right insets).
  • Example 12 IFN ⁇ R1 d39 Prevents Upregulation of MHCs in ES Cells
  • IFN ⁇ Activated T-cells secrete IFN ⁇ , which binds to the IFN ⁇ R1/R2 complex expressed on many cell types, including tissues and cells derived from ES cells. IFN ⁇ binding to the IFN ⁇ receptor induces upregulation of HLA (MHC in mice) and HLA-related molecules on the cell surface, which increases the allogenicity of the allograft and the likelihood of immune rejection. Differences in HLA proteins (also called major antigens) between the donor and recipient are the primary cause of rejection in all allogenic transplants.
  • transfected C57BL/6 ES cells with piggyback-integratable vectors containing a wild-type IFN ⁇ R1 or dominant negative IFN ⁇ R1 (IFN ⁇ R1 d39, which lacks 39 amino acids in the cytoplasmic tail) transgene. These transgenes were expressed under the control of a constitutive CAG promoter upstream of the transgene contained on the same piggyback-integrated cassette.
  • IFN ⁇ R1 d39 dominant negative IFN ⁇ R1
  • Wild type and transfected ES cells were then grown in culture and exposed to 100 ng/mL of IFN ⁇ ligand for 24 hours.
  • IFN ⁇ exposure resulted in increased expression of the H-2kb and H-2Db major histocompatibility surface molecules (MHC class I), but not in IFN ⁇ R1 d39 cells (right panel of FIG. 20 ).
  • Exposure to PBS alone had no effect.
  • MHC class I levels were detected by fluorescent antibody staining, and the expression level quantified by measuring the mean fluorescent intensity (MFI) by flow cytometry.
  • IFN ⁇ R1 d39 is a useful immunosuppressive transgene that can be expressed by the cloaked cells described herein to reduce immune activation and transplant rejection.
  • Example 13 Administration of Cloaked Cells Expressing a VEGF Inhibitor to a Subject with Wet AMD
  • a physician of skill in the art can treat a patient, such as a human patient, with wet AMD to reduce vascularization of the eye or prevent or reduce disease progression.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked RPE cells or cloaked stem cells that have been differentiated into RPE cells) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG) and a VEGF inhibitor (e.g., VEGF-Trap, e.g., aflibercept) under the control of a constitutive promoter (e.g.,
  • the cloaked cells may be administered to the patient, for example, by local administration to the eye (e.g., injection into the subretinal space), to treat wet AMD. Twenty five thousand to one hundred thousand cloaked cells (e.g., 25,000, 50,000, 75,000, or 100,000 cloaked cells) can be administered to each affected eye.
  • a practitioner of skill in the art can monitor the expression of the VEGF inhibitor, and the patient's improvement in response to the therapy, by a variety of methods. For example, a physician can monitor the patient's vision and the vascularization of the eye using standard approaches. A finding that the patient's vision improves or does not worsen, or that vascularization of the eye decreases or does not worsen compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • Example 14 Administration of Cloaked Dopaminergic Neurons to a Subject with Parkinson's Disease (PD)
  • a physician of skill in the art can treat a patient, such as a human patient, with PD to reduce motor symptoms of PD (e.g., bradykinesia, tremors, or rigidity) or prevent or reduce disease progression.
  • PD motor symptoms of PD
  • a physician of skill in the art can treat a patient, such as a human patient, with PD to reduce motor symptoms of PD (e.g., bradykinesia, tremors, or rigidity) or prevent or reduce disease progression.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., dopaminergic neurons that have been modified to express cloaking transgenes or cloaked stem cells that have been differentiated into dopaminergic neurons) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG).
  • a constitutive promoter e.g., CMV or CAG
  • the cloaked cells may be administered to the patient, for example, by local administration to the central nervous system (e.g., stereotactic injection into the substantia nigra), to treat PD. Twenty five thousand to one hundred thousand cloaked cells (e.g., 25,000, 50,000, 75,000, or 100,000 cloaked cells) can be administered.
  • the patient can optionally be administered an additional therapy for PD, such as a dopamine agonist.
  • a practitioner of skill in the art can monitor the patient's improvement in response to the therapy by a variety of methods. For example, a physician can monitor the patient's movement using standard neurological tests. A finding that the patient's motor symptoms improve or do not worsen compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • Example 15 Administration of Cloaked Cardiac Muscle Cells to a Subject that has Suffered a Myocardial Infarction
  • a physician of skill in the art can treat a patient, such as a human patient, who has recently suffered a myocardial infarction to improve cardiac function (e.g., to replace or dead or damaged cardiac muscle cells).
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked cardiac muscle cells or cloaked stem cells that have been differentiated into cardiac muscle cells) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG).
  • a constitutive promoter e.g., CMV or CAG
  • the cloaked cells may be administered to the patient, for example, by local administration to the heart (e.g., injection into the cardiac muscle), to promote recovery after the myocardial infarction.
  • the cells can be injected into the cardiac muscle as a monotherapy, or the cells can be delivered during the performance of a bypass surgery or another open heart surgical procedure.
  • One million to five billion cloaked cardiac muscle cells can be administered.
  • 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 cloaked cells) can be administered.
  • a practitioner of skill in the art can monitor the patient's improvement in response to the therapy by a variety of methods. For example, a physician can monitor the patient's cardiac function using standard approaches (e.g., EKG, echocardiogram, angiogram, stress test, or nuclear imaging). A finding that the patient's cardiac function improves or stabilizes compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • standard approaches e.g., EKG, echocardiogram, angiogram, stress test, or nuclear imaging.
  • Example 16 Administration of Cloaked Cells Expressing a TNF ⁇ Inhibitor to a Subject with RA
  • a physician of skill in the art can treat a patient, such as a human patient, with rheumatoid arthritis to reduce join stiffness, swelling, or pain.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked articular fibroblasts or cloaked stem cells that have been differentiated into articular fibroblasts) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG) and a TNF ⁇ inhibitor (e.g., a TNF ⁇ inhibitory antibody, such as adalimumab) under the control of an
  • the cloaked cells may be administered to the patient, for example, by local administration to a joint (e.g., injection into an arthritic joint, such as joint in the hand), to treat RA.
  • a joint e.g., injection into an arthritic joint, such as joint in the hand
  • One million to one hundred million cloaked articular fibroblasts expressing an anti-inflammatory biologic e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8 cloaked articular fibroblasts) can be administered to each affected joint.
  • an anti-inflammatory biologic e.g., 1 ⁇ 10
  • the patient When the patient experiences a flare up of RA symptoms, the patient can be treated with tetracycline or doxycycline to drive expression of the TNF ⁇ inhibitor. Tetracycline or doxycycline can be withdrawn when the patient's flare up has resolved.
  • a practitioner of skill in the art can monitor the expression of the TNF ⁇ inhibitor, and the patient's improvement in response to the therapy, by a variety of methods. For example, a physician can monitor the patient's joint pain, swelling, and stiffness using standard approaches. A finding that the patient's joint pain, swelling, or stiffness is reduced compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • Example 17 Administration of Cloaked Cells Expressing Insulin to a Subject with Type 1 Diabetes
  • a physician of skill in the art can treat a patient, such as a human patient, with Type 1 diabetes to increase insulin levels.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked stem cells, cloaked pancreatic beta cells, or cloaked stem cells that have been differentiated into pancreatic beta cells) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG) and insulin under the control of a constitutive promoter (e.g., CMV or CAG).
  • a constitutive promoter e.g., CMV or CAG
  • insulin e.g., CMV or CA
  • the cloaked cells may be administered to the patient, for example, by subcutaneous injection (e.g., to create a cloaked subcutaneous tissue), to treat Type 1 diabetes.
  • subcutaneous injection e.g., to create a cloaked subcutaneous tissue
  • One million to three billion cloaked cells expressing insulin e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇
  • a practitioner of skill in the art can monitor the expression of the patient's improvement in response to the therapy by a variety of methods. For example, a physician can monitor insulin levels or symptoms of Type 1 diabetes (e.g., unintended weight loss, fatigue, or blurred vision) using standard approaches. A finding that the patient's insulin levels are increased or the symptoms of Type 1 diabetes are reduced compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • Type 1 diabetes e.g., unintended weight loss, fatigue, or blurred vision
  • Example 18 Administration of Cloaked Cells Expressing Factor VIII to a Subject with Hemophilia
  • a physician of skill in the art can treat a patient, such as a human patient, with hemophilia to increase the levels of a blood clotting factor or reduce excessive bleeding or bruising.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked stem cells, cloaked endothelial cells, or cloaked stem cells that have been differentiated into endothelial cells) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG) and Factor VIII under the control of a constitutive promoter (e.g., CMV or CAG).
  • a constitutive promoter e.
  • the cloaked cells may be administered to the patient, for example, by subcutaneous injection (e.g., to create a cloaked subcutaneous tissue), to treat hemophilia.
  • subcutaneous injection e.g., to create a cloaked subcutaneous tissue
  • One million to three billion cloaked cells expressing Factor VIII e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 6
  • a practitioner of skill in the art can monitor the expression of the patient's improvement in response to the therapy by a variety of methods. For example, a physician can monitor Factor VIII levels or symptoms of hemophilia (e.g., excessive bleeding or frequent bruising) using standard approaches. A finding that the patient's Factor VIII levels are increased or the symptoms of hemophilia are reduced compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • Factor VIII levels or symptoms of hemophilia e.g., excessive bleeding or frequent bruising
  • Example 19 Administration of Cloaked Cells Expressing Glucocerebrosidase to a Subject with Gaucher's Disease
  • a physician of skill in the art can treat a patient, such as a human patient, with Gaucher's disease to reduce the accumulation of glucocerebroside or to reduce symptoms of Gaucher's disease (e.g., fatigue, anemia, low blood platelet count, enlarged liver or spleen).
  • a patient such as a human patient
  • Gaucher's disease e.g., fatigue, anemia, low blood platelet count, enlarged liver or spleen.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked stem cells) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG) and glucocerebrosidase under the control of a constitutive promoter (e.g., CMV or CAG).
  • a constitutive promoter e.g., CMV or CAG
  • glucocerebrosidase under the control of a constitutive promoter (e.g., CMV or CAG).
  • the cloaked cells may be administered to the patient, for example, by subcutaneous injection (e.g., to create a cloaked subcutaneous tissue), to treat Gaucher's disease.
  • subcutaneous injection e.g., to create a cloaked subcutaneous tissue
  • One million to three billion cloaked cells expressing glucocerebrosidase e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10
  • a practitioner of skill in the art can monitor the expression of the patient's improvement in response to the therapy by a variety of methods. For example, a physician can monitor accumulation of glucocerebroside or symptoms of Gaucher's disease (e.g., fatigue, anemia, low blood platelet count, enlarged liver or spleen) using standard approaches. A finding of a reduction in the patient's accumulation of glucocerebroside or symptoms of Gaucher's disease compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • Gaucher's disease e.g., fatigue, anemia, low blood platelet count, enlarged liver or spleen
  • Example 20 Administration of Cloaked Cells to a Subject Receiving a Liver Transplant
  • a physician of skill in the art can treat a patient, such as a human patient, who is receiving a liver transplant to reduce the risk of transplant rejection.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked stem cells, cloaked liver cells, or cloaked stem cells that have been differentiated into liver cells) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG).
  • a constitutive promoter e.g., CMV or CAG
  • the cloaked cells may be administered to the patient, for example, by injection into the liver or near the site of the transplanted liver, to reduce the risk of transplant rejection.
  • One million to one hundred billion cloaked cells e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , 3 ⁇ 10 9 , 4 ⁇ 10 9 , 5 ⁇ 10 9 , 6 ⁇ 10 8
  • a practitioner of skill in the art can monitor the expression of the patient's improvement in response to the therapy by a variety of methods. For example, a physician can monitor the patient for symptoms that predict transplant rejection using standard approaches. A finding of an equivalent outcome in transplant rejection as that observed in subjects administered immunosuppressive agent(s) indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • Example 21 Administration of Cloaked and Fail Safe Cells Expressing Insulin to a Subject with Type 1 Diabetes
  • a physician of skill in the art can treat a patient, such as a human patient, with Type 1 diabetes to increase insulin levels.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked stem cells, cloaked pancreatic beta cells, or cloaked stem cells that have been differentiated into pancreatic beta cells) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG) and insulin under the control of a constitutive promoter (e.g., CMV or CAG).
  • a constitutive promoter e.g., CMV or CAG
  • insulin e.g., CMV or CA
  • the cloaked cells can also be modified to allow for control of their proliferation by linking the expression of a CDL with that of a DNA sequence encoding a negative selectable marker.
  • the cloaked cells can be modified to contain homozygous ALINKS (e.g., HSV-TK systems) in two CDL loci (e.g., Cdk1 and Top2A).
  • the cloaked cells may be administered to the patient, for example, by subcutaneous injection (e.g., to create a cloaked subcutaneous tissue), to treat Type 1 diabetes.
  • One million to three billion cloaked cells expressing insulin can be administered subcutaneously.
  • insulin e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , or 3 ⁇ 10 9 cloaked cells
  • a practitioner of skill in the art can monitor the expression of the patient's improvement in response to the therapy by a variety of methods. For example, a physician can monitor insulin levels or symptoms of Type 1 diabetes (e.g., unintended weight loss, fatigue, or blurred vision) using standard approaches. A finding that the patient's insulin levels are increased or the symptoms of Type 1 diabetes are reduced compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed.
  • Type 1 diabetes e.g., unintended weight loss, fatigue, or blurred vision
  • a practitioner of skill in the art can also monitor the size of the cloaked subcutaneous tissue. If it appears that the cloaked subcutaneous tissue is becoming tumorigenic, the practitioner can administer ganciclovir to the subject to ablate the proliferating cloaked cells. Non-proliferating cloaked cells will not express the CDLs, and, therefore, will not be ablated by ganciclovir treatment.
  • Example 22 Administration of Cloaked and Fail Safe Cells Expressing Insulin to a Subject with Type 1 Diabetes
  • a physician of skill in the art can treat a patient, such as a human patient, with Type 1 diabetes to increase insulin levels.
  • a physician of skill in the art can administer to the human patient cloaked cells (e.g., cloaked stem cells, cloaked pancreatic beta cells, or cloaked stem cells that have been differentiated into pancreatic beta cells) that express one or more (e.g., one, two, three, four, five, six, seven, or all eight) of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8, and Serpin B9 (Spi6) under the control of a constitutive promoter (e.g., CMV or CAG) and insulin under the control of a constitutive promoter (e.g., CMV or CAG).
  • a constitutive promoter e.g., CMV or CAG
  • insulin e.g., CMV or CA
  • the cloaked cells can also be modified to allow for control of their proliferation by linking the expression of a CDL with that of a DNA sequence encoding an inducible activator system.
  • a dox-bridge can be inserted into two CDLs (e.g., Cdk1 and Top2A) to generate homozygous modifications in both CDLs in a cloaked cell, such that in the presence of an inducer (e.g., doxycycline) the dox-bridge permits CDL expression, thereby allowing cell division and proliferation.
  • the cloaked cells may be administered to the patient, for example, by subcutaneous injection (e.g., to create a cloaked subcutaneous tissue), to treat Type 1 diabetes.
  • One million to three billion cloaked cells expressing insulin can be administered subcutaneously.
  • insulin e.g., 1 ⁇ 10 6 , 2 ⁇ 10 6 , 3 ⁇ 10 6 , 4 ⁇ 10 6 , 5 ⁇ 10 6 , 6 ⁇ 10 6 , 7 ⁇ 10 6 , 8 ⁇ 10 6 , 9 ⁇ 10 6 , 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 ,4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 , or 1 ⁇ 10 8 , 2 ⁇ 10 8 , 3 ⁇ 10 8 , 4 ⁇ 10 8 , 5 ⁇ 10 8 , 6 ⁇ 10 8 , 7 ⁇ 10 8 , 8 ⁇ 10 8 , 9 ⁇ 10 8 , 1 ⁇ 10 9 , 2 ⁇ 10 9 , or 3 ⁇ 10 9 cloaked cells
  • a practitioner of skill in the art can monitor the expression of the patient's improvement in response to the therapy by a variety of methods. For example, a physician can monitor insulin levels or symptoms of Type 1 diabetes (e.g., unintended weight loss, fatigue, or blurred vision) using standard approaches. A finding that the patient's insulin levels are increased or the symptoms of Type 1 diabetes are reduced compared to measurements taken prior to administration of the cloaked cells indicates that the patient is responding favorably to the treatment. Subsequent doses can be determined and administered as needed. If the practitioner determines that the subject needs a higher level of insulin, the practitioner can allow the cloaked cells to proliferate by treating the subject with doxycycline. Once the desired level of insulin is reached, treatment with doxycycline can be stopped and the cloaked cells will cease to proliferate.
  • Type 1 diabetes e.g., unintended weight loss, fatigue, or blurred vision
  • CDLs (ID refers to EntrezGene identification number; CS score refers to the CRISPR score average provided in Wang et al., 2015; function refers to the known or predicted function the locus, of predictions being based on GO terms, as set forth in the Gene Ontology Consortium website http://geneontology.org/; functional category refers to 4 categories of cell functions based on the GO term-predicted function; CDL (basis) refers to information that the inventors used to predict that a gene is a CDL, predictions being based on CS score, available gene knockout (KO) data, gene function, and experimental data provided in WO 2016 141480).
  • ID refers to EntrezGene identification number
  • CS score refers to the CRISPR score average provided in Wang et al., 2015
  • function refers to the known or predicted function the locus, of predictions being based on GO terms, as set forth in the Gene Ontology Consortium website http://geneontology.org/
  • functional category refers to 4
  • Name ID CS Function Functional CDL (mouse) (mouse) (human) (human) score (GO term) category (basis)
  • Anapc2 99152 ANAPC2 29882 ⁇ 2.88 mitotic cell cycle Cell cycle CS score Wirth K G, et al.
  • DNA repair DNA CS score DNA- replication
  • replication function DNA repair Nol11 68979 NOL11 25926 ⁇ 1.59 transcription, DNA- DNA CS score, templated replication, function DNA repair Nol8 70930 NOL8 55035 ⁇ 1.35 DNA replication DNA CS score, replication, function DNA repair Pcna 18538 PCNA 5111 ⁇ 3.60 DNA replication DNA CS score, Roa S, et al. Proc replication, mouse Natl Acad Sci USA. DNA repair K.O., 2008 Oct.
  • DNA- DNA CS score dependent DNA replication, function replication DNA repair Pold2 18972 POLD2 5425 ⁇ 2.51 DNA replication DNA CS score, replication, function DNA repair Pole 18973 POLE 5426 ⁇ 2.90 DNA replication DNA CS score, replication, function DNA repair Polr1a 20019 POLR1A 25885 ⁇ 2.62 transcription, DNA- DNA CS score, templated replication, function DNA repair n/a n/a POLR2J2 246721 ⁇ 3.08 transcription, DNA- DNA CS score, templated replication, function DNA repair Polr3a 218832 POLR3A 11128 ⁇ 2.43 transcription, DNA- DNA CS score, templated replication, function DNA repair Polr3c 74414 POLR3C 10623 ⁇ 2.02 transcription, DNA- DNA CS score, templated replication, function DNA repair Polr3h 78929 POLR3H 171568 ⁇ 2.66
  • RNA CS score for protein translation tran- function scription, protein translation Cdc5l 71702 CDC5L 988 ⁇ 2.09 mRNA splicing, via RNA CS score, spliceosome tran- function scription, protein translation Cdc73 214498 CDC73 79577 ⁇ 2.58 negative regulation of RNA CS score, Wang P, et al.
  • RNA polymerase II scription K.O., May; 28(9): 2930-40 promoter protein function translation Cebpz 12607 CEBPZ 10153 ⁇ 2.11 transcription from RNA CS score, RNA polymerase II tran- function promoter scription, protein translation Clasrp 53609 CLASRP 11129 ⁇ 1.30 mRNA processing RNA CS score, tran- function scription, protein translation Clp1 98985 CLP1 10978 ⁇ 3.47 mRNA splicing, via RNA CS score, Hanada T, et al. spliceosome tran- mouse Nature. 2013 Mar.
  • RNA CS score from RNA tran- function polymerase II scription, promoter protein translation Cpsf1 94230 CPSF1 29894 ⁇ 2.58 mRNA splicing, via RNA CS score, spliceosome tran- function scription, protein translation Cpsf2 51786 CPSF2 53981 ⁇ 2.55 mRNA RNA CS score, polyadenylation tran- function scription, protein translation Cpsf3l 71957 CPSF3L 54973 ⁇ 2.09 snRNA processing RNA CS score, tran- function scription, protein translation Dars 226414 DARS 1615 ⁇ 2.90 translation RNA CS score, tran- function scription, protein translation Dbr1 83703 DBR1 51163 ⁇ 3.75 RNA splicing, via RNA CS score, spliceosome tran- function scription, protein translation Cpsf2 51786 CPSF2 53981 ⁇ 2.55 mRNA RNA
  • RNA polymerase II scription K.O., 2010 promoter protein function April; 151(4): 1948-58 translation Ears2 67417 EARS2 124454 ⁇ 1.91 tRNA aminoacylation RNA CS score, for protein translation tran- function scription, protein translation Ebna1bp2 69072 EBNA1BP2 10969 ⁇ 1.52 ribosome biogenesis RNA CS score, tran- function scription, protein translation Eef1a1 13627 EEF1A1 1915 ⁇ 3.11 translational RNA CS score, elongation tran- function scription, protein translation Eef1g 67160 EEF1G 1937 ⁇ 1.42 translation RNA CS score, tran- function scription, protein translation Eef2 13629 EEF2 1938 ⁇ 3.53 translation RNA CS score, tran- function scription, protein translation Eftud2 20624 EFTUD2 9343 ⁇ 3.79 mRNA splicing, via RNA CS score,
  • RNA CS score tRNA 3′-trailer RNA CS score, cleavage, tran- function endonucleolytic scription, protein translation Ell 13716 ELL 8178 ⁇ 2.23 transcription RNA CS score, Mitani K, et al. elongation from RNA tran- mouse Biochem Biophys polymerase II scription, K.O., Res Commun. 2000 promoter protein function Dec.
  • RNA polymerase I tran- mouse Biochem Biophys promoter scription K.O., Res Commun. 2008 protein function Jan. 25; 365(4): 636- translation 42 Polr1c 20016 POLR1C 9533 ⁇ 2.79 transcription from RNA CS score, RNA polymerase I tran- function promoter scription, protein translation Polr2a 20020 POLR2A 5430 ⁇ 3.15 transcription from RNA CS score, RNA polymerase II tran- function promoter scription, protein translation Polr2b 231329 POLR2B 5431 ⁇ 3.09 transcription from RNA CS score, RNA polymerase II tran- function promoter scription, protein translation Polr2c 20021 POLR2C 5432 ⁇ 3.15 mRNA splicing, via RNA CS score, spliceosome tran- function scription, protein translation Polr2d 69241 POLR2D 5433 ⁇ 2.23 nuclear- RNA CS score, transcribed mRNA tran-
  • RNA CS score synthesis of RNA tran- function primer scription, protein translation Prpf38b 66921 PRPF38B 55119 ⁇ 2.68 mRNA processing RNA CS score, tran- function scription, protein translation Prpf4 70052 PRPF4 9128 ⁇ 2.24 RNA splicing RNA CS score, tran- function scription, protein translation Prpf8 192159 PRPF8 10594 ⁇ 3.43 mRNA splicing, via RNA CS score, spliceosome tran- function scription, protein translation Ptcd1 71799 PTCD1 26024 ⁇ 1.77 tRNA 3′-end RNA CS score, processing tran- function scription, protein translation Pwp2 110816 PWP
  • RNA CS score protein function translation Sf3a1 67465 SF3A1 10291 ⁇ 3.18 mRNA 3′-splice site RNA CS score, recognition tran- function scription, protein translation Sf3a2 20222 SF3A2 8175 ⁇ 2.66 mRNA 3′-splice site RNA CS score, recognition tran- function scription, protein translation Sf3a3 75062 SF3A3 10946 ⁇ 2.26 mRNA splicing, via RNA CS score, transesterification tran- function reactions scription, protein translation Sf3b2 319322 SF3B2 10992 ⁇ 2.51 mRNA splicing, via RNA CS score, spliceosome tran- function scription, protein translation Sf3b3 101943 SF3B3 23450 ⁇ 4.13 RNA splicing, via RNA
  • RNA polymerase II scription K.O., 2005 Jul. promoter protein function 1; 19(13): 581-95 translation Smg5 229512 SMG5 23381 ⁇ 2.35 nuclear- RNA CS score, transcribed mRNA tran- function catabolic process, scription, nonsense- protein mediated decay translation Smg6 103677 SMG6 23293 ⁇ 1.18 nuclear- RNA CS score, transcribed mRNA tran- function catabolic process, scription, nonsense- protein mediated decay translation Snrnp25 78372 SNRNP25 79622 ⁇ 2.43 mRNA processing RNA CS score, tran- function scription, protein translation Snrnp27 66618 SNRNP27 11017 ⁇ 1.36 mRNA processing RNA CS score, tran- function scription, protein translation Snrpd2 107686 SNRPD2 6633 ⁇ 2.47 RNA splicing RNA CS score, tran- function scription

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11459372B2 (en) 2020-11-30 2022-10-04 Crispr Therapeutics Ag Gene-edited natural killer cells
US11473060B2 (en) 2020-12-30 2022-10-18 Crispr Therapeutics Ag Compositions and methods for differentiating stem cells into NK cells
US11492591B2 (en) 2015-05-08 2022-11-08 President And Fellows Of Harvard College Universal donor stem cells and related methods
US11566230B2 (en) 2020-12-31 2023-01-31 Crispr Therapeutics Ag Universal donor cells
WO2023023278A3 (en) * 2021-08-18 2023-06-08 University Of Utah Research Foundation Multigene constructs for treatment of age-related macular degeneration and other complement dysregulation-related conditions
WO2024091959A1 (en) * 2022-10-24 2024-05-02 The Regents Of The University Of California Drug resistant immune cells

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10704021B2 (en) 2012-03-15 2020-07-07 Flodesign Sonics, Inc. Acoustic perfusion devices
CA2935960C (en) 2014-01-08 2023-01-10 Bart Lipkens Acoustophoresis device with dual acoustophoretic chamber
US11377651B2 (en) 2016-10-19 2022-07-05 Flodesign Sonics, Inc. Cell therapy processes utilizing acoustophoresis
US11708572B2 (en) 2015-04-29 2023-07-25 Flodesign Sonics, Inc. Acoustic cell separation techniques and processes
BR112020009889A2 (pt) 2017-12-14 2020-11-03 Flodesign Sonics, Inc. acionador e controlador de transdutor acústico
US20220016181A1 (en) * 2018-12-13 2022-01-20 Sinai Health System Immunomodulatory cells and uses thereof
CN110179824A (zh) * 2019-04-01 2019-08-30 普米斯生物技术(珠海)有限公司 一种通过免疫调控治疗ii型糖尿病的新方法
US11162079B2 (en) 2019-05-10 2021-11-02 The Regents Of The University Of California Blood type O Rh-hypo-immunogenic pluripotent cells
US20220307013A1 (en) * 2019-08-30 2022-09-29 The Regents Of The University Of California Gene fragment overexpression screening methodologies, and uses thereof
CN116234906A (zh) 2020-01-13 2023-06-06 萨那生物技术股份有限公司 血型抗原的修饰
EP4103725A4 (en) * 2020-02-14 2024-03-27 Asklepios Biopharmaceutical Inc METHOD FOR TREATING GENE THERAPY-ASSOCIATED TOXICITY WITH ANTIBIOTICS
US20230149461A1 (en) * 2020-04-01 2023-05-18 Nanjing Legend Biotech Co., Ltd. Compositions and methods for reducing graft rejection in allogeneic cell therapy
MX2022012792A (es) * 2020-04-30 2023-02-14 Res Institute At Nationwide Children´S Hospital Superar la inmunosupresion con celulas nk resistentes a tgf-\03b2.
CN112089847B (zh) * 2020-08-31 2021-05-28 高连如 一种融合pnv靶向缺血心肌血管的干细胞药物及制备方法
CN112972681B (zh) * 2021-01-27 2022-06-07 西安交通大学 Mt-nd6作为新靶点在代谢综合征诊断和治疗药物中的应用
US20230016422A1 (en) * 2021-06-23 2023-01-19 Crispr Therapeutics Ag Engineered cells with improved protection from natural killer cell killing
WO2023098729A1 (en) * 2021-11-30 2023-06-08 Biocytogen Pharmaceuticals (Beijing) Co., Ltd. Genetically modified non-human animal with human or chimeric genes
CN117384851A (zh) * 2022-07-11 2024-01-12 士泽生物医药（苏州）有限公司 一种表达faslg的通用型细胞及其制备方法
CN116785312B (zh) * 2023-06-21 2023-11-14 天津医科大学眼科医院 miR-15a-5p在治疗眼底疾病中的应用

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1879448A2 (en) * 2005-02-25 2008-01-23 University of Chicago Compositions and methods related to serpin spi6
PE20110020A1 (es) * 2005-10-13 2011-01-31 Anthrogenesis Corp Inmunomodulacion mediante el uso de celulas madres de la placenta
EP2547328B1 (en) * 2010-02-11 2017-06-07 Ecole Polytechnique Federale de Lausanne (EPFL) Ccr7 ligand delivery and co-delivery in immunotherapy
CN104619722B (zh) * 2012-06-22 2022-10-04 达特茅斯大学理事会 新型vista-ig构建体和vista-ig用于治疗自身免疫性、过敏性和炎性疾病的用途
EP3483178B1 (en) * 2012-07-31 2020-10-28 AgeX Therapeutics, Inc. Methods to produce hla-g-modified cells
EP3021942A4 (en) * 2013-07-19 2017-04-19 The Regents of The University of California Milk fat globule epidermal growth factor 8 regulates fatty acid uptake
EP3480213B1 (en) * 2014-06-18 2019-11-13 Albert Einstein College of Medicine Syntac polypeptides and uses thereof
US11319555B2 (en) * 2014-11-20 2022-05-03 Duke University Compositions, systems and methods for cell therapy
FI3850946T3 (fi) * 2014-12-05 2023-12-28 Regeneron Pharma Ei-ihmiseläimiä, joilla on humanisoitu erilaistumisklusterin 47 geeni
LT3268480T (lt) * 2015-03-09 2021-07-26 Sinai Health System Priemonės ir būdai, skirti panaudoti ląstelių dalijimosi lokusus ląstelių proliferacijos kontroliavimui
CN107533051B (zh) * 2015-03-27 2020-11-13 南加利福尼亚大学 Hla-g作为car t细胞免疫疗法的新靶标
US11207393B2 (en) * 2015-10-16 2021-12-28 President And Fellows Of Harvard College Regulatory T cell PD-1 modulation for regulating T cell effector immune responses

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Bhattacharya et al. (2017, PLoS ONE 12(3): e0171355, https://doi.org/10.1371/journal.pone.0171355) *
Fenton et al., 2020, Medicinal Chemistry Research 29:1133-1146 *
Tokuriki et al., 2009, Curr. Opin. Struc. Biol. 19:596-604 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11492591B2 (en) 2015-05-08 2022-11-08 President And Fellows Of Harvard College Universal donor stem cells and related methods
US11618881B2 (en) 2015-05-08 2023-04-04 President And Fellows Of Harvard College Universal donor stem cells and related methods
US11459372B2 (en) 2020-11-30 2022-10-04 Crispr Therapeutics Ag Gene-edited natural killer cells
US11591381B2 (en) 2020-11-30 2023-02-28 Crispr Therapeutics Ag Gene-edited natural killer cells
US11473060B2 (en) 2020-12-30 2022-10-18 Crispr Therapeutics Ag Compositions and methods for differentiating stem cells into NK cells
US11566230B2 (en) 2020-12-31 2023-01-31 Crispr Therapeutics Ag Universal donor cells
US11578309B2 (en) 2020-12-31 2023-02-14 Crispr Therapeutics Ag Universal donor cells
WO2023023278A3 (en) * 2021-08-18 2023-06-08 University Of Utah Research Foundation Multigene constructs for treatment of age-related macular degeneration and other complement dysregulation-related conditions
WO2024091959A1 (en) * 2022-10-24 2024-05-02 The Regents Of The University Of California Drug resistant immune cells

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