Allograft tolerance without the need for systemic immune suppression

Abstract

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, and methods for making and using the same are provided. The cell comprises a set of transgenes, each transgene encoding a gene product that is cytoplasmic, membrane bound, or local acting, and whose function is one or more of: to mitigate antigen presenting cell activation and function; to mitigate graft attacking leukocyte activity or cytolytic function; to mitigate macrophage cytolytic function and phagocytosis of allograft cells; to induce apoptosis in graft attacking leukocytes; to mitigate local inflammatory proteins; and to protect against leukocyte-mediated apoptosis. The transgenes comprise two or more of the following genes: PD-L 1, HLAG or H2-M3, Cd47, Cd200, FASLG or Fasl, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6.

1 . A cell genetically modified to comprise
 a set of transgenes comprising two or more of the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6, or comprising a gene encoding a biologic that acts as an agonist of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, or Serpin B9 or Spi6.   
     
     
         2 . The cell of  claim 1 , wherein the set of transgenes comprises three, four, five, six, seven, or all eight of the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6. 
     
     
         3 . The cell of  claim 1 , wherein the set of transgenes gone comprises PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6. 
     
     
         4 . The cell of  claim 1 , further comprising:
 (a) one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1 r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39 or a gene encoding a biologic that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39;   (b) at least one mechanism for controlling cell proliferation, the genetically modified cell comprising a polynucleotide encoding:
 i) a negative selectable marker that is inserted into a locus of a gene, wherein, optionally, the gene is an endogenous essential gene, the expression of which is necessary for cell viability or proliferation, wherein the cell co-expresses the negative selectable marker and the gene product encoded by the gene, and wherein, in the presence of an inducer of the negative selectable marker, the negative selectable marker promotes the killing of the cell; and/or 
 ii) an exogenous inducible transcriptional activator inserted into a locus of a gene, wherein, optionally, the gene is an endogenous essential gene, the expression of which is necessary for cell viability or proliferation, wherein the exogenous inducible transcriptional activator regulates expression of the gene, and wherein, in the absence of an inducer of the exogenous inducible transcriptional activator, the gene is not expressed, thereby promoting the killing of the cell or inhibiting proliferation of the cell; and/or 
   (c) a transgene encoding a therapeutic agent.   
     
     
         5 . The cell of  claim 4 , wherein:
 (a) the TGF-β or biologic is local acting in the graft environment   (b) the genetic modification of the locus of the gene is a homozygous, heterozygous, hemizygous or compound heterozygous genetic modification and/or wherein the genetic modification of the locus of the gene results in expression of the gene only in the presence of an inducer of the exogenous inducible transcriptional activator;   (c) the gene is one or more of the genes recited in Table 5;   (d) the gene product functions in one or more of cell cycle regulation, DNA replication, RNA transcription, protein translation, and metabolism;   (e) the gene is one, two, or more of CDK1, TOP2A, CENPA, BIRC5, and EEF2;   (f) the negative selectable marker comprises herpes simplex virus-thymidine kinase, cytosine deaminase, carboxyl esterase, or iCasp9;   (q) the exogenous inducible transcriptional activator and the inducer form a system, wherein the system comprises a doxycycline-inducible system, a cumate switch inducible system, an ecdysone inducible system, a radio wave inducible system, or a ligand-reversible dimerization system;   (h) the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 17;   (i) the therapeutic agent is a protein or an antibody;   (j) the therapeutic agent is an agent listed in Table 2 or the wild-type version of a gene known to be mutated in cancer, an enzyme or hormone deficiency, a metabolic disorder, or a degenerative disease;   (k) 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; or   (l) the therapeutic agent is expressed using a constitutive promoter selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1a 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.   
     
     
         6 . The cell of  claim 1 , wherein:
 (a) the cell is a stem cell, a cell amenable for genome editing, and/or a source of a therapeutic cell type;   (b) one or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 is expressed at a level that is greater than the expression level of the corresponding endogenous gene in the cell or that is equal to or greater than the expression level of the corresponding endogenous gene in an activated leukocyte;   (c) the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12;   (d) the HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 15;   (e) the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4;   (f) the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6;   (g) the FASLG or FasL transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 9;   (h) the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1;   (i) the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14;   (j) the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 7;   (k) the two or more transgenes is operably linked to a constitutive promoter; and/or   (l) one or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 is expressed at a level that is in the top 5% of gene expression for all genes in the genome of the cell.   
     
     
         7 . The cell of  claim 6 , wherein:
 (a) the cell is an embryonic stem cell, a pluripotent stem cell, an induced pluripotent stem cell (iPSC), 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 hepatoblast, a myoblast, a preadipocyte, a hepatocyte, a chondrocyte, a smooth muscle cell, a K562 human erythroid leukemia cell line, a bone cell, a synovial cell, a tendon cell, a ligament cell, a meniscus cell, an adipose cell, a dendritic cell, a natural killer cell, a skeletal muscle cell, a cardiac muscle cell, an erythroid-megakaryocytic cell, an eosinophil, a macrophage, a T cell, an islet beta-cell, a neuron, a cardiomyocyte, a blood cell, an exocrine progenitor, a ductal cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a PP cell, a cholangiocyte, a white or brown adipocyte, a hormone-secreting cell, an epidermal keratinocyte, an epithelial cell, a kidney cell, a germ cell, a skeletal joint synovium cell, a periosteum cell, a perichondrium cell, a cartilage cell, an endothelial cell, a pericardium cell, a meningeal cell, a keratinocyte precursor cell, a keratinocyte stem cell, a pericyte, a glial cell, an ependymal cell, a cell isolated from an amniotic or placental membrane, a serosal cell, a somatic cell, or a cell derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach;   (b) all eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 are expressed at a level that is greater than the expression level of the corresponding endogenous gene in the cell or that is equal to or greater than the expression level of the corresponding endogenous gene in an activated leukocyte;   (c) the constitutive promoter is selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1a 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; or   (d) all eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 are expressed at a level that is in the top 5% of gene expression for all genes in the genome of the cell.   
     
     
         8 .- 30 . (canceled) 
     
     
         31 . The cell of claim  540 , wherein:
 (a) the antibody is an inhibitory antibody, or agonist antibody;   (b) the negative selectable marker comprises herpes simplex virus-thymidine kinase and the inducer comprises ganciclovir;   (c) the negative selectable marker comprises cytosine deaminase and the inducer comprises 5-fluorocytosine;   (d) the negative selectable marker comprises carboxyl esterase and the inducer comprises irinotecan; or   (e) the negative selectable marker comprises iCasp9 and the inducer comprises AP1903.   
     
     
         32 .- 35 . (canceled) 
     
     
         36 . A method for providing a local immunosuppression at a transplant site in an allogeneic host, the method comprising transplanting the cell of  claim 1  or a population of said cell at a transplantation site in an allogeneic host. 
     
     
         37 . (canceled) 
     
     
         38 . The method of  claim 36 , wherein the set of transgenes comprises three, four, five, six, seven, or all eight of the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6. 
     
     
         39 . The method of  claim 36 , wherein the set of transgenes genes comprises PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6. 
     
     
         40 . The method of  claim 36 , further comprising expressing one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1 r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, and IFNγR1 d39 or a gene encoding a biologic that acts as an agonist of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1, or IFNγR1 d39. 
     
     
         41 . The method of  claim 40 , wherein:
 (a) the TGF-β or biologic is local acting in the graft environment; and/or   (b) the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 17.   
     
     
         42 . (a) the cell is a stem cell, a cell amenable for genome editing, and/or a source of a therapeutic cell type;
 (b) one or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 is expressed at a level that is greater than the expression level of the corresponding endogenous gene in the cell or that is equal to or greater than the expression level of the corresponding endogenous gene in an activated leukocyte;   (c) the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12;   (d) the HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 15;   (e) the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4;   (f) the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6;   (g) the FASLG or FasL transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 9;   (h) the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1;   (i) the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14;   (j) the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 7;   (k) the two or more transgenes is operably linked to a constitutive promoter;   (l) the cell further comprises a transgene encoding a therapeutic agent; and/or   (m) one or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 is expressed at a level that is in the top 5% of gene expression for all genes in the genome of the cell.   
     
     
         43 . The method of  claim 42 , wherein:
 (a) the cell is an embryonic stem cell, an induced pluripotent stem cell, an induced pluripotent stem cell (iPSC), 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 hepatoblast, a myoblast, a preadipocyte, a hepatocyte, a chondrocyte, a smooth muscle cell, a K562 human erythroid leukemia cell line, a bone cell, a synovial cell, a tendon cell, a ligament cell, a meniscus cell, an adipose cell, a dendritic cell, a natural killer cell, a skeletal muscle cell, a cardiac muscle cell, an erythroid-megakaryocytic cell, an eosinophil, a macrophage, a T cell, an islet beta-cell, a neuron, a cardiomyocyte, a blood cell, an exocrine progenitor, a ductal cell, an acinar cell, an alpha cell, a beta cell, a delta cell, a PP cell, a cholangiocyte, a white or brown adipocyte, a hormone-secreting cell, an epidermal keratinocyte, an epithelial cell, a kidney cell, a germ cell, a skeletal joint synovium cell, a periosteum cell, a perichondrium cell, a cartilage cell, an endothelial cell, a pericardium cell, a meningeal cell, a keratinocyte precursor cell, a keratinocyte stem cell, a pericyte, a glial cell, an ependymal cell, a cell isolated from an amniotic or placental membrane, a serosal cell, a somatic cell, or a cell derived from skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine, or stomach;   (b) all eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 are expressed at a level that is greater than the expression level of the corresponding endogenous gene in the cell or that is equal to or greater than the expression level of the corresponding endogenous gene in an activated leukocyte;   (c) the constitutive promoter is selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1a 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   (d) the therapeutic agent is a protein or antibody;   (e) the therapeutic agent is an agent listed in Table 2 or the wild-type version of a gene that is mutated in the subject;   (f) 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; or   (g) the therapeutic agent is expressed using a constitutive promoter selected from the group consisting of the CAG promoter, the cytomegalovirus (CMV) promoter, the EF1a 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; or   (h) all eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8, and Serpin B9 or Spi6 are expressed at a level that is in the top 5% of gene expression for all genes in the genome of the cell.   
     
     
         44 . A method of controlling proliferation of the cell of  claim 1  at a transplant site in an allogeneic host, the method comprising:
 a) genetically modifying a locus of a gene of the cell, wherein, optionally, the gene is an endogenous essential gene, the expression of which is necessary for cell viability or proliferation, the genetic modification of the locus of the gene comprising one or more of:
 i) polynucleotide encoding a negative selectable marker that is inserted into the locus of the gene such that the cell co-expresses the negative selectable marker and the gene product encoded by the gene; and 
 ii) a polynucleotide encoding an exogenous inducible transcriptional activator inserted into the locus of the gene such that the exogenous inducible transcriptional activator promotes expression of the gene upon exposure of the cell to an inducer of the exogenous inducible transcriptional activator; 
 
 b) transplanting the cell of step (a) or a population of the cells of step (a) at a transplantation site in an allogeneic host; and 
 c) i) permitting proliferation of the transplanted cell comprising the negative selectable marker by maintaining the transplanted cell comprising the negative selectable marker in the absence of an inducer of the negative selectable marker or ablating and/or inhibiting proliferation of the transplanted cell comprising the negative selectable marker by exposing the cell comprising the negative selectable marker to the inducer of the negative selectable marker; and/or 
 ii) permitting proliferation of the transplanted cell comprising the exogenous inducible transcriptional activator by exposing the transplanted cell comprising the exogenous inducible transcriptional activator to an inducer of the exogenous inducible transcriptional activator or preventing or inhibiting proliferation of the transplanted cell comprising the exogenous inducible transcriptional activator by maintaining the cell comprising the exogenous inducible transcriptional activator in the absence of the inducer of the exogenous inducible transcriptional activator. 
 
     
     
         45 . The method of  claim 36 , wherein:
 (a) the allogeneic host is a mammal; and/or   (b) the host has degenerative disease or condition that can be treated with cell therapy.   
     
     
         46 . The method of  claim 45 , wherein:
 (a) the allogeneic host is a mouse or a human; and/or   (b) the disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, an enzyme or hormone deficiency, a metabolic disorder, an autoimmune disease, age-related macular degeneration, retinal dystrophy, an infectious disease, hemophilia, myocardial infarction, a degenerative disease, or an age-related disease.   
     
     
         47 .- 64 . (canceled) 
     
     
         65 . The method of  claim 43 , wherein the antibody is an inhibitory antibody or agonist antibody. 
     
     
         66 .- 68 . (canceled) 
     
     
         69 . A composition comprising the cell of  claim 1  or a plurality of said cell. 
     
     
         70 . The composition of  claim 69 , further comprising a pharmaceutically acceptable excipient. 
     
     
         71 . A kit comprising the composition of  claim 69 . 
     
     
         72 . A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject the composition of  claim 69 . 
     
     
         73 . The method of  claim 72 , wherein:
 (a) the disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, a lung disease, a blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, an enzyme or hormone deficiency, a metabolic disorder, an autoimmune disease, age-related macular degeneration, retinal dystrophy, an infectious disease, hemophilia, myocardial infarction, a degenerative disease, an age-related disease, or a disease or condition listed in Table 2;   (b) the cells are differentiated into a lineage restricted cell type prior to administration to the subject;   (c) the cells are administered locally to the tissue or body site in need of cells or the therapeutic agent;   (d) the cells are administered intravenously, subcutaneously, intramuscularly, percutaneously, intradermally, parenterally, intraarterially, intravascularly, or by perfusion;   (e) the cells are administered as a tissue;   (f) the cells are administered in an amount of 25,000 to 5,000,000,000 cells;   (g) the method further comprises administering an additional therapeutic agent;   (h) the method further comprises controlling proliferation of the cell; or   (i) the cell is removed after completion of the therapy.   
     
     
         74 . (canceled) 
     
     
         75 . The method of claim  7374 , wherein:
 (a) the disease or condition is myocardial infarction and the cells are differentiated into cardiac muscle cells,   (b) the disease or condition is blindness and the cells are differentiated into photoreceptor cells;   (c) the disease or condition is spinal cord injury, Parkinson's disease, Huntington's disease, Alzheimer's disease and the cells are differentiated into neurons;   (d) the disease or condition is multiple sclerosis and the cells are differentiated into glial cells;   (e) the cells are administered by subcutaneous injection to produce a cloaked subcutaneous tissue;   (f) the tissue is administered with a gel, biocompatible matrix, or cellular scaffold;   (g) the cells are administered in an amount of 800,000,000 to 3,000,000,000 cells;   (h) the additional therapeutic agent is administered prior to administration of the cells, after administration of the cells, or concurrently with administration of the cells;   (i) 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, an anti-histamine, Anti-TNFα, azathioprine, belimumab, beta interferon, a calcineurin inhibitor, certolizumab, a corticosteroid, cromolyn, cyclosporin A, cyclosporine, dimethyl fumarate, etanercept, fingolimod, fumaric acid esters, glatiramer acetate, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonist, long-acting beta2 agonist, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, a probiotic, a retinoid, salicylic acid, short-acting beta2 agonist, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab, or vedolizumab, bevacuzimab, ranibizumab, or aflibercept), photodynamic therapy, photocoagulation, carbidopa-levodopa, a dopamine agonist, an MAO-B inhibitor, a catechol-O-methyltransferase inhibitor, an anticholinergic, amantadine, deep brain stimulation, an anticoagulant, an anti-platelet agent, an angiotensin-converting enzyme inhibitor, an angiotensin II receptor blocker, an angiotensin receptor neprilysin inhibitor, a beta blocker, a combined alpha and beta blocker, a calcium channel blocker, a cholesterol lowering medication, a nicotinic acid, a cholesterol absorption inhibitor, a digitalis preparation, a diuretic, a vasodilator, a dual anti-platelet therapy, a cardiac procedure, an antiviral compound, a nucleoside-analog reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor, an antibacterial compound, an antifungal compound, an antiparasitic compound, insulin, a sulfonylurea, a biguanide, a meglitinide, a thiazolidinedione, a DPP-4 inhibitor, an SGLT2 inhibitor, an alpha-glucosidase inhibitor, a bile acid sequestrant, aspirin, a dietary regimen, a clotting factor, desmopressin, a clot-preserving medication, a fibrin sealant, physical therapy, a coenzyme, a bone marrow transplant, an organ transplant, hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium-chain triacylglycerols, miglustat, enzyme supplementation therapy, a checkpoint inhibitor, a chemotherapeutic drug, a biologic drug, radiation therapy, cryotherapy, hyperthermia, surgical excision or tumor tissue, or an anti-cancer vaccine;   (j) the cell comprises a negative selectable marker, wherein, optionally, the negative selectable marker is inserted at a locus of an endogenous essential gene, the expression of which is necessary for cell viability or proliferation, and the method of controlling proliferation comprises:
 i) permitting proliferation of the cell comprising the negative selectable marker by maintaining the cell comprising the negative selectable marker in the absence of an inducer of the negative selectable marker; or 
 ii) ablating or inhibiting proliferation of the cell comprising the negative selectable marker by exposing the cell comprising the negative selectable marker to the inducer of the negative selectable marker; or 
   (k) the cell comprises an exogenous inducible transcriptional activator, wherein, optionally, the exogenous inducible transcriptional activator is inserted at a locus of an endogenous essential gene, the expression of which is necessary for cell viability or proliferation, and the method of controlling cell proliferation comprises:
 i) permitting proliferation of the cell comprising the exogenous inducible transcriptional activator by exposing the cell comprising the exogenous inducible transcriptional activator to an inducer of the exogenous inducible transcriptional activator; or 
 ii) preventing or inhibiting proliferation of the cell comprising the exogenous inducible transcriptional activator by maintaining the cell comprising the exogenous inducible transcriptional activator in the absence of the inducer of the exogenous inducible transcriptional activator. 
   
     
     
         76 .- 192 . (canceled)