Genetic modification of personalized regenerative cells

Abstract

Means, methods, and compositions of matter for gene modifying autologous personalized regenerative cells and derivatives thereof. Patient somatic cells can be dedifferentiated to pluripotent stem cells and said pluripotent stem cells or derivatives thereof are gene modified in order to provide a therapeutic benefit. Autologous mesenchymal or hematopoietic stem cells, or tissue specific progenitor cells can be modified.

Description

IMMORTA-GMPRC-PCT GENETIC MODIFICATION OF PERSONALIZED REGENERATIVE CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims the benefit of priority to United States Provisional Application Serial No.63 / 615,296, filed on December 28, 2023, the entire contents of which are incorporated herein by reference. FIELD OF THE INVENTION

[0002] The invention pertains to the field of therapeutics in regenerative medicine, more particularly, the invention relates to the field of genetically modified stem cells, more particularly the invention pertains to the use of inducible constructs to manipulate personalized regenerative cell stem cells into inducing specific biological responses at a desired time and location. BACKGROUND

[0003] Mesenchymal stem cells (MSCs) are the formative pluripotential blast cells found inter alia in bone marrow, blood, dermis and periosteum that are capable of differentiating into more than one specific type of mesenchymal or connective tissue (i.e., the tissues of the body that support the specialized elements, e.g., adipose, osseous, stroma, cartilaginous, elastic and fibrous connective tissues) depending upon various influences from bioactive factors, such as cytokines. The potential to differentiate into cells such as osteoblasts and chondrocytes is retained after isolation and expansion in culture; differentiation occurs when the cells are induced in vitro under specific conditions or placed in vivo at the site of damaged tissue.

[0004] At present sources of mesenchymal stem cells are limited. Furthermore, there are no methods of manipulating mesenchymal stem cells genetically to repair damaged tissue through continual replenishment using immortal sources. SUMMARY

[0005] Disclosed are means, methods, and compositions of matter for gene modifying autologous personalized regenerative cells and derivatives thereof. In one embodiment patient somatic cells are dedifferentiated to pluripotent stem cells and saidIMMORTA-GMPRC-PCT pluripotent stem cells or derivatives thereof are gene modified to provide a therapeutic benefit. In one embodiment autologous mesenchymal or hematopoietic stem cells are modified. In other embodiments tissue specific progenitor cells are modified.

[0006] The invention provides means of gene modifying personalized regenerative cells and derivatives thereof for use in gene therapy. Embodiments of the invention provide methods for modifying a reprogrammed autologous cell for use in gene therapy, wherein the reprogrammed autologous cell is derived from a personalized regenerative cell (e.g., using a differentiation protocol). Specifically, the invention provides the use of inducible genetic elements to selectively upregulate or downregulate gene expression in these cells.

[0007] In one embodiment of the invention, an in vitro reprogrammed autologous cell comprising a vector is provided, wherein the vector comprises a polynucleotide, and wherein the polynucleotide comprises a gene switch, said gene switch comprising the following: a) at least one transcription factor sequences, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and wherein said ligand-dependent transcription factor comprises an ecdysone receptor ligand binding domain, operably linked to a promoter; and b) a polynucleotide encoding a polypeptide linked to a promoter that is activated by the ligand-dependent transcription factor. In one embodiment, the polypeptide is at least 85% identical to the wild-type human therapeutic polypeptide sequence. In certain embodiments, a therapeutic polypeptide sequence is selected based on the desired therapeutic effect of the engineered cell.

[0008] In one embodiment, the in vitro reprogrammed autologous cell is administered to a mammal with a disease (preferably a human), and a first administration of a ligand to the mammal is administered less than 48 hours after the cells are administered, and wherein the ligand is administered daily for a period of 2 to 30 days. In certain embodiments, the mammal exhibits antitumor immunity after said population of in vitro engineered cells and said ligand are administered to said mammal.

[0009] In one aspect, the in vitro reprogrammed autologous cell comprises a vector selected from a group of vectors comprising of: a) a lentiviral vector; b) adenoviral vector; c) an adeno-associated viral vector. In one aspect, the vector comprises aIMMORTA-GMPRC-PCT polynucleotide encoding a gene switch that comprises a first transcription factor sequence and a second transcription factor sequence under the control of a promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. In certain embodiments, the first transcription factor and the second transcription factor are connected by an internal ribosomal entry site.

[0010] In certain embodiments, an in vitro reprogrammed autologous cell utilized to practice the invention is generated from peripheral blood cells possessing enhanced regenerative potential as compared to peripheral blood mononuclear cells, wherein enhanced regenerative potential is quantified by expression of one or a plurality of biomarkers comprising CD133, c-met, or CD34. In other embodiments, enhanced regenerative potential is quantified by placing the in vitro reprogrammed cell in 1% oxygen for at least 2 hours and quantifying IL-3 production in culture.

[0011] In one embodiment, an in vitro reprogrammed autologous cell comprises a vector, wherein the vector comprises a polynucleotide encoding a gene switch, and wherein the polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor.

[0012] In a specific embodiment, a reprogrammed autologous cell comprises a vector, wherein the vector comprises a polynucleotide encoding a gene switch, and wherein the polynucleotide encoding a gene switch comprises the following: (1) a first transcription factor sequence encoding a VP-16 transactivation domain and a chimeric RXR ligand binding domain; (2) an EMCV IRES; and (3) a second transcription factor sequence encoding a GAL4 DNA-binding domain and a Choristoneura fumiferana ligand binding domain comprising a substitution mutation; wherein said ligand is a diacylhydrazine that is administered once daily for 14 consecutive days beginning on the day that said in vitro engineered cells are administered.

[0013] Embodiments of the invention provide polynucleotides encoding therapeutic molecules that are useful for endowing therapeutic properties to a cell generated using theIMMORTA-GMPRC-PCT methods of the invention. In certain embodiments, a therapeutic cell comprising a reprogrammed autologous cell is transfected with one or a plurality of polynucleotides, wherein the one or plurality of polynucleotide sequences encode therapeutic polynucleotides.

[0014] In one embodiment, a reprogrammed autologous cell is transfected with a polynucleotide gene construct encoding an anti-apoptotic gene. In one embodiment, the anti-apoptotic gene is selected from the group comprising obestatin, XIAP, sphingosine- 1-phosphate, BCL-2, BCL-XL, GATA-4, IGF-1, EGF, heme-oxygenase-1, NF-kB, akt, pi3-k, and epha-2.

[0015] In one embodiment, a reprogrammed autologous cell is transfected with a polynucleotide gene construct capable of inducing RNA interference directed against a molecule associated with induction of apoptosis. In one embodiment, the molecule associated with induction of apoptosis is selected from the group comprising Fas, FasL, CASP1 (ICE), CASP10 (MCH4), CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR (CASPER), CRADD, PYCARD (TMS1 / ASC), ABL1, AKT1, BAD, BAK1, BAX, BCL2L11, BCLAF1, BID, BIK, BNIP3, BNIP3L, CASP1 (ICE), CASP10 (MCH4), CASP14, CASP2, CASP4, CASP6, CASP8, CD70 (TNFSF7), CIDEB, CRADD, FADD, FASLG (TNFSF6), HRK, LTA (TNFB), NOD1 (CARD4), PYCARD (TMS1 / ASC), RIPK2, TNF, TNFRSF10A, TNFRSF10B (DR5), TNFRSF25 (DR3), TNFRSF9, TNFSF10 (TRAIL), TNFSF8, TP53, TP53BP2, TRADD, TRAF2, TRAF3, and TRAF4.

[0016] In one embodiment, a reprogrammed autologous cell is induced to differentiate into a neural cell lineage. In certain embodiments, differentiation into the neural cell lineage is conferred by transfecting the cell with a polynucleotide gene construct encoding a polypeptide selected from the group comprising DCYAP1R1, ARTN, BDNF, CD40 (TNFRSF5), CNTF, CNTFR, CRHBP, CRHR1, CRHR2, FRS2, FRS3, FUS, GDNF, GFRA1, GFRA2, GFRA3, GMFB, GMFG, MAGED1, MT3, NF1, NGF, NGFR, NGFRAP1, NR1I2, NRG1, NRG2, NTF3, NTF4, NTRK1, NTRK2, PSPN, PTGER2, TFG, TRO, and VGF.

[0017] In another embodiment, a reprogrammed autologous cell is endowed with the ability to migrate or home to a tissue or a tumor site. In certain embodiments,IMMORTA-GMPRC-PCT migration or homing ability is conferred by transfecting the reprogrammed autologous cell with a polynucleotide gene construct encoding a polypeptide selected from the group comprising SDF-1, VEGF, FGF-1, FGF-2, PDGF-BB, angiopoietin, and TSPL-1.

[0018] In another embodiment, a reprogrammed autologous cell is endowed with the ability to stimulate angiogenesis. In certain embodiments, angiogenic ability is conferred by transfecting the reprogrammed autologous cell with a polynucleotide gene construct encoding a polypeptide selected from the group comprising activin A, adrenomedullin, aFGF, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4, bFGF, B61, bFGF inducing activity, cadherins, CAM-RF, cGMP analogs, ChDI, CLAF, claudins, collagen, collagen receptors .alpha..sub.1.beta..sub.1 and .alpha..sub.2.beta..sub.1, connexins, Cox-2, ECDGF (endothelial cell-derived growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, endothelins, endostatin, endothelial cell growth inhibitor, endothelial cell-viability maintaining factor, endothelial differentiation sphingolipid G-protein coupled receptor-1 (EDG1), ephrins, Epo, HGF, TGF-beta, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin and fibronectin receptor .alpha.5.beta.1, Factor X, HB-EGF, HBNF, HGF, HUAF, heart derived inhibitor of vascular cell proliferation, IL1, IGF-2 IFN-gamma, integrin receptors, K-FGF, LIF, leiomyoma-derived growth factor, MCP-1, macrophage-derived growth factor, monocyte-derived growth factor, MD-ECI, MECIF, MMP 2, MMP3, MMP9, urokinase plasminogen activator, neuropilin (NRP1, NRP2), neurothelin, nitric oxide donors, nitric oxide synthases (NOSs), notch, occludins, zona occludins, oncostatin M, PDGF, PDGF-B, PDGF receptors, PDGFR-.beta., PD- ECGF, PAI-2, PD-ECGF, PF4, P1GF, PKR1, PKR2, PPAR-gamma, PPAR-gamma ligands, phosphodiesterase, prolactin, prostacyclin, protein S, smooth muscle cell-derived growth factor, smooth muscle cell-derived migration factor, sphingosine-1-phosphate-1 (SIP1), Syk, SLP76, tachykinins, TGF-beta, Tie 1, Tie2, TGF-.beta., and TGF-.beta. receptors, TIMPs, TNF-alphatransferrin, thrombospondin, urokinase, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF.sub.164, VEGI, and EG-VEGF.

[0019] In yet another embodiment, a reprogrammed autologous cell is endowed with the ability to stimulate an immune response in a subject such an anti-tumor response. In certain embodiments, immune stimulatory ability is conferred by transfecting the reprogrammed autologous cell with a polynucleotide gene construct encoding aIMMORTA-GMPRC-PCT polypeptide selected from the group comprising ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, CD40LG (TNFSF5), IFNA2, , IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL-6, IL8, IL9, IL-18, IL-33, LTA, LTB, MIF, SCYE1, SPP1, TNF, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), C5, CCL1 (I- 309), CCL11 (eotaxin), HMGB1, IL-2. IL-12, IL-17, IL33. CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), complement component C3, complement component C5, 2,3 alpha gal, CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK) , CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC / IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9, IL13, and IL8. DETAILED DESCRIPTION

[0020] The detailed description set forth below is intended as a description of various configurations of the subject technology and is not intended to represent the only configurations in which the subject technology may be practiced. The detailed description includes specific details for the purpose of providing an understanding of the subject technology. It will be apparent to those skilled in the art that the subject technology may be practiced without these specific details.

[0021] Unless defined otherwise, the technical terms used herein have the same meaning as is commonly understood by one of skill in the art.

[0022] As used herein, the terms “subject” or “patient” refers to a human or another mammal to whom a method or therapy of the invention is applied.

[0023] As used herein, “induced pluripotent stem cell” (“iPSC”) or “personalized regenerative cell” refers to a pluripotent stem cell that can be reprogrammed from adult somatic cells (e.g., from skin, from peripheral blood cells, from bone marrow, or from another tissue source). For example, an induced pluripotent stem cell may be reprogrammed by the introduction of specific genes encoding transcription factors Oct3 / 4, Sox2, c-Myc and Klf4 into mouse adult fibroblasts under embryonic stem (ES)IMMORTA-GMPRC-PCT cell culture conditions. A personalized regenerative cell may refer to a pluripotent stem cell that is amenable to modification or differentiation to provide an autologous therapeutic cell for treating the same subject. For example, in certain embodiments, a personalized progenitor cell is used to generate an autologous cell that is amenable to gene modification using the methods of the invention, wherein the autologous cell may comprise a progenitor cell, a tissue-specific progenitor cell, a multipotent cell, a mesenchymal cell, a hematopoietic cell, a fibroblast, a lymphocyte, or another cell type.

[0024] As used herein, “reprogramming” when used in the context of a cell refers to a process that uses reprogramming factors such as transcription factors to convert one cell type into another cell type. For example, “reprogramming” may comprise conversion of a somatic cell into an induced pluripotent stem cell. Reprogramming of a cell may involve introducing reprogramming factors into a cell type through genetic manipulations and / or using chemical compounds.

[0025] As used herein, “cancer” is used to describe an uncontrolled growth of cells. The term “anti-cancer” is intended to encompass not only the treatment of a cancer disease, that is, inhibition of proliferation of cancer cells or cancer stem cells, or elimination of cancer cells or cancer stem cells, but also prevention of a cancer disease, that is, improvement of resistance to cancer prior to the onset of cancer. Thus, the term “prevention or treatment of cancer” or “inhibition of cancer” and “anti-cancer” are used interchangeably.

[0026] As used herein, the terms “marker" refers to a gene expression product that is differentially present in a sample taken from two different subjects, e.g., from a test subject or patient having a disease or event, compared to a comparable sample taken from a control subject. In some contexts, these terms refer to a gene expression product that is differentially present in a subject, cell, tissue, or organ relative to another subject, cell, tissue, or organ.

[0027] As used herein, “inhibitors,” “activators,” and “modulators” of expression or of activity are used to refer to inhibiting, activating, or modulating cells, respectively, and are identified using in vitro and in vivo assays for expression or activity. The term “modulator” includes inhibitors and activators. A modulator can be an antibody or a soluble ligand which binds a protein of interest. Inhibitors are agents that, e.g., inhibitIMMORTA-GMPRC-PCT expression of a polypeptide or polynucleotide of the invention or bind to, partially or totally block stimulation or enzymatic activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of a polypeptide or polynucleotide of the invention, e.g., antagonists. Preferred modulators according to the invention, inhibit or suppress immune responses to an antigen or alloantigen. Assays to identify inhibitors and activators include, e.g., applying putative modulators to immune cells and then determining the functional effects of the cell on the immune response. Inhibitors or modulators are compared to control samples without the inhibitor or modulator to examine the extent of effect. Control samples (untreated with modulators) are assigned a relative activity value of 100%. Inhibition is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control sample is about 80%, optionally 50% or 25 to 1%, or less. Activation is achieved when the activity value of a polypeptide or polynucleotide of the invention relative to the control sample is 110%, optionally 150%, optionally 200-500%, or 1000-3000%, or higher.

[0028] As used herein, the term “isolated” when used with regard to a population of cells refers to a cell population which either has no naturally occurring counterpart or has been separated or purified from other components, including other cell types, which naturally accompany it, e.g., in normal or diseased tissues such as lung, kidney, or placenta, tumor tissue such as colon cancer tissue, or body fluids such as blood, serum, or urine. Typically, an isolated cell population is enriched at least two-fold, at least four- fold, at least eight-fold, or more for a specified cell type when compared to the natural source from which the population was obtained.

[0029] As used herein, “mesenchymal stem cell” or “MSC” refers to cells that are (1) adherent to plastic; and (2) express CD73, CD90, and CD105 antigens, while being CD14, CD34, CD45, and HLA-DR negative, and (3) possess ability to differentiate to osteogenic, chondrogenic and adipogenic lineage. As used herein, “mesenchymal stromal cell” or “MSC” can be derived from any tissue including, but not limited to, bone marrow, adipose tissue, amniotic fluid, endometrium, trophoblast-derived tissues, cord blood, Wharton jelly, placenta, amniotic tissue, derived from pluripotent stem cells, and tooth. As used herein, “mesenchymal stromal cell” or “MSC” includes cells that are CD34 positive upon initial isolation from tissue but are similar to cells described about phenotypically and functionally. As used herein, “MSC” includes cells that are isolatedIMMORTA-GMPRC-PCT from tissues using cell surface markers selected from the list comprised of NGF-R, PDGF-R, EGF-R, IGF-R, CD29, CD49a, CD56, CD63, CD73, CD105, CD106, CD140b, CD146, CD271, MSCA-1, SSEA4, STRO-1 and STRO-3 or any combination thereof and satisfy the ISCT criteria either before or after expansion. As used herein, “mesenchymal stromal cell” or “MSC” includes cells described in the literature as bone marrow stromal stem cells (BMSSC), marrow-isolated adult multipotent inducible cells (MIAMI) cells, multipotent adult progenitor cells (MAPC), mesenchymal adult stem cells (MASCS), MultiStem®, Prochymal®, remestemcel-L, Mesenchymal Precursor Cells (MPCs), Dental Pulp Stem Cells (DPSCs), PLX cells, PLX-PAD, AlloStem®, Astrostem®, Ixmyelocel-T, MSC-NTF, NurOwn™, Stemedyne™-MSC, Stempeucel®, StempeucelCLI, StempeucelOA, HiQCell, Hearticellgram-AMI, Revascor®, Cardiorel®, Cartistem®, Pneumostem®, Promostem®, Homeo-GH, AC607, PDA001, SB623, CX601, AC607, Endometrial Regenerative Cells (ERC), adipose-derived stem and regenerative cells (ADRCs).

[0030] As used herein, "angiogenesis" refers to any alteration of an existing vascular bed or the formation of new vasculature which benefits tissue perfusion. This includes the formation of new vessels by sprouting of endothelial cells from existing blood vessels or the remodeling of existing vessels to alter size, maturity, direction or flow properties to improve blood perfusion of tissues.

[0031] As used herein, "therapeutically effective amount" refers to the quantity of cells, conditioned media, or exosomes that, when administered to a mammal for treating a condition, symptom, or complication is sufficient to affect such treatment. The "therapeutically effective amount" may vary depending on the disease, stage, and symptoms, as well as on the characteristics and responsiveness of the mammal to be treated.

[0032] As used herein, DNA "coding sequence" refers to a double-stranded DNA sequence that encodes a polypeptide and can be transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of suitable regulatory sequences. "Suitable regulatory sequences" refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences mayIMMORTA-GMPRC-PCT include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0033] As used herein, “open reading frame" is abbreviated ORF and refers to a length of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0034] As used herein, "head-to-head" describes the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-head orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 5' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds away from the 5' end of the other polynucleotide. The term "head-to-head" may be abbreviated (5')-to-(5') and may also be indicated by the symbols (.rarw..fwdarw.) or (3'.rarw.5'5'.fwdarw.3').

[0035] As used herein, “tail-to-tail" describes the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a tail-to-tail orientation when the 3' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds toward the other polynucleotide. The term "tail-to-tail" may be abbreviated (3')-to-(3') and may also be indicated by the symbols (.fwdarw..rarw.) or (5'.fwdarw.3'3.rarw.5').

[0036] As used herein, "head-to-tail" is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-tail orientation when the 5' end of the coding strand of one polynucleotide is adjacent to the 3' end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds in the sameIMMORTA-GMPRC-PCT direction as that of the other polynucleotide. The term "head-to-tail" may be abbreviated (5')-to-(3') and may also be indicated by the symbols (.fwdarw..fwdarw.) or (5'.fwdarw.3'5'.fwdarw.3').

[0037] As used herein, "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0038] As used herein, "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0039] As used herein, "restriction endonuclease" and "restriction enzyme" are used interchangeably and refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.

[0040] As used herein, “therapeutic agent” means to have "therapeutic efficacy" in modulating angiogenesis and / or wound healing / and / or treatment of a pathology through augmentation of a desired therapeutic effect. In a specific embodiment the amount of the therapeutic is said to be an "angiogenic modulatory amount", if administration of that amount of the therapeutic is sufficient to cause a significant modulation (i.e., increase or decrease) in angiogenic activity when administered to a subject (e.g., an animal model or human patient) needing modulation of angiogenesis.

[0041] As used herein, "growth factor" may refer to a naturally occurring, endogenous or exogenous protein, or recombinant protein, capable of stimulating cellular proliferation and / or cellular differentiation and cellular migration. A growth factor may comprise any protein, polypeptide, variant or portion thereof that is capable of, directly or indirectly, including endothelial cell growth. Such proteins include, for example, acidic and basic fibroblast growth factors (aFGF) (GenBank Accession No. NP.sub.--149127) and bFGF (GenBank Accession No. AAA52448), vascular endothelial growth factor (VEGF) (GenBank Accession No. AAA35789 or NP.sub.--001020539), epidermalIMMORTA-GMPRC-PCT growth factor (EGF) (GenBank Accession No. NP.sub.--001954), transforming growth factor .alpha. (TGF-.alpha.) (GenBank Accession No. NP.sub.--003227) and transforming growth factor .beta. (TFG-.beta.) (GenBank Accession No.1109243A), platelet-derived endothelial cell growth factor (PD-ECGF) (GenBank Accession No. NP.sub.--001944), platelet-derived growth factor (PDGF) (GenBank Accession No.1109245A), tumor necrosis factor .alpha. (TNF-.alpha.) (GenBank Accession No. CAA26669), hepatocyte growth factor (HGF) (GenBank Accession No. BAA14348), insulin like growth factor (IGF) (GenBank Accession No. P08833), erythropoietin (GenBank Accession No. P01588), colony stimulating factor (CSF), macrophage-CSF (M-CSF) (GenBank Accession No. AAB59527), granulocyte / macrophage CSF (GM-CSF) (GenBank Accession No. NP.sub.--000749), monocyte chemotactic protein-1 (GenBank Accession No. P13500) and nitric oxide synthase (NOS) (GenBank Accession No. AAA36365). See, Klagsbrun, et al., Annu. Rev. Physiol., 53:217-239 (1991); Folkman, et al., J. Biol. Chem., 267:10931-10934 (1992) and Symes, et al., Current Opinion in Lipidology, 5:305-312 (1994). Variants or fragments of a mitogen may be used as long as they induce or promote endothelial cell or endothelial progenitor cell growth. Preferably, the endothelial cell mitogen contains a secretory signal sequence that facilitates secretion of the protein. Proteins having native signal sequences, e.g., VEGF, are preferred. Proteins that do not have native signal sequences, e.g., bFGF, can be modified to contain such sequences using routine genetic manipulation techniques. See, Nabel et al., Nature, 362:844 (1993).

[0042] As used herein, "expression vector" refers to a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence following transformation into the host. The cloned gene, i.e., the inserted nucleic acid sequence, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like. Initiation control regions or promoters, which are useful to drive expression of a nucleic acid in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving expression of these genes can be used in an expression vector, including but not limited to, viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue specific promoters, pathogenesis or disease related promoters, developmental specific promoters, inducible promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1,IMMORTA-GMPRC-PCT TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichia); .beta.-lactamase, lac, ara, tet, trp, lP.sub.L, lP.sub.R, T7, tac, and trc promoters (useful for expression in Escherichia coli); light regulated-, seed specific-, pollen specific-, ovary specific-, cauliflower mosaic virus 35S, CMV 35S minimal, cassava vein mosaic virus (CsVMV), chlorophyll a / b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase, stress inducible, rice tungro bacilliform virus, plant super- promoter, potato leucine aminopeptidase, nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells); animal and mammalian promoters known in the art including, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, a baculovirus 1E1 promoter, an elongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, .alpha.-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis or disease related-promoters, and promoters that exhibit tissue specificity and have been utilized in transgenic animals, such as the elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region active in pancreatic beta cells, immunoglobulin gene control region active in lymphoid cells, mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells; albumin gene, Apo AI and Apo AII control regions active in liver, alpha- fetoprotein gene control region active in liver, alpha 1-antitrypsin gene control region active in the liver, beta-globin gene control region active in myeloid cells, myelin basic protein gene control region active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region active in skeletal muscle, and gonadotropic releasing hormone gene control region active in the hypothalamus, pyruvate kinase promoter, villin promoter, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell .alpha.-actin, and the like. In addition, these expression sequences may be modified by addition of enhancer or regulatory sequences and the like.IMMORTA-GMPRC-PCT

[0043] As used herein, "vector" refers to any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term "vector" includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. Possible vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector. Another example of vectors that are useful in the invention is the UltraVector.TM. Production System (Intrexon Corp., Blacksburg, VA) as described in WO 2007 / 038276. For example, the insertion of the DNA fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the DNA molecules may be enzymatically modified, or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini. Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome. Such markers allow identification and / or selection of host cells that incorporate and express the proteins encoded by the marker. Viral vectors, and particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, adeno- associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. In addition to a nucleic acid, a vector may also comprise one or more regulatory regions, and / or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).

[0044] The invention provides methods of gene modifying personalized regenerative cells and derivatives thereof for use in gene therapy. Specifically, the invention provides the use of inducible genetic elements to selectively upregulate orIMMORTA-GMPRC-PCT downregulate gene expression in a cell of the invention. In certain embodiments, a cell of the invention that is subjected to genetic modification comprises a reprogrammed autologous cell that is derived or differentiated from an induced pluripotent stem cell, wherein the reprogrammed autologous cell may comprise a progenitor cell, a tissue- specific progenitor cell, a multipotent cell, a mesenchymal cell, a hematopoietic cell, a fibroblast, a lymphocyte, or another cell type depending on the culture conditions provided. In certain embodiments, a reprogrammed autologous cell, by virtue of being derived from an induced pluripotent stem cell, has different phenotypic or functional characteristics as compared to a cell of the same type or lineage that is isolated directly from a tissue source of a subject. In certain embodiments, an in vitro reprogrammed autologous cell is transfected with one or a plurality of polynucleotides / gene constructs encoding therapeutic polypeptides using the methods of the invention (e.g., a polynucleotide encoding a molecule that stimulates angiogenesis or an immune response), or a cell that has not undergone modifications of gene expression.

[0045] Various aspects of the invention are enumerated in the following paragraphs:

[0046] Aspect 1: A method for treating a subject with an in vitro reprogrammed autologous cell, the method comprising: a) identifying a subject with a disease such as cancer; b) providing an in vitro reprogrammed autologous cell, wherein the cell is transfected with a vector, wherein the vector comprises a polynucleotide encoding a gene switch, and wherein the gene switch comprises the following: i) at least one transcription factor sequence that encodes a ligand-dependent transcription factor comprising an ecdysone receptor ligand binding domain, operably linked to a promoter; and ii) a polynucleotide encoding a therapeutic polypeptide sequence linked to a promoter that is activated by said ligand-dependent transcription factor, wherein the therapeutic polypeptide sequence is at least 85% identical to the wild type human polypeptide sequence; c) administering the in vitro reprogrammed autologous cell to the subject; and d) administering the ligand to the subject. In certain embodiments, a polynucleotide encodes a therapeutic polypeptide sequence that comprises a molecule, wherein the molecule is involved in a cellular function comprising angiogenesis, immune suppression, immune stimulation, homing or chemotaxis, or differentiation into a particular cell lineage, e.g., neural, adipocyte, mesenchymal, lymphocytic, endothelial, hepatic, osteogenic, pancreatic, myocytic, cardiac, or others. In certain embodiments, a cellIMMORTA-GMPRC-PCT comprising a therapeutic polypeptide sequence is administered to a subject for treating a disease such as cancer.

[0047] Aspect 2: The method of Aspect 1, wherein the ligand is administered to the subject less than 48 hours after administering the reprogrammed autologous cell.

[0048] Aspect 3: The method of Aspect 1, wherein the ligand is administered to the subject daily for a period of 2 to 30 days following administration of the reprogrammed autologous cell.

[0049] Aspect 4: The method of Aspect 1, wherein the ligand is administered to the subject daily for a period of at least 2 days, at least 7 days, at least 10 days, at least 14 days, or at least 30 days beginning on the day that the reprogrammed autologous cells are administered to the subject.

[0050] Aspect 5: The method of Aspect 3, wherein administration of the ligand increases the therapeutic effects of the administered reprogrammed autologous cell in the subject.

[0051] Aspect 6: The method of Aspect 1, wherein the polynucleotide encoding a gene switch comprises a first transcription factor sequence and a second transcription factor sequence under the control of the same promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor.

[0052] Aspect 7: The method of Aspect 5, wherein the first transcription factor and the second transcription factor are connected by an internal ribosomal entry site.

[0053] Aspect 8: The method of Aspect 1, wherein the reprogrammed autologous cell is generated by isolating a somatic cell from the subject, dedifferentiating the somatic cell into an induced pluripotent stem cell (iPSC), and subsequently differentiating the iPSC into a reprogrammed autologous therapeutic cell.

[0054] Aspect 9: The method of Aspect 8, wherein the somatic cell is a peripheral blood mononuclear cell.IMMORTA-GMPRC-PCT

[0055] Aspect 10: The method of Aspect 9, wherein the peripheral blood mononuclear cell possesses enhanced regenerative potential as compared to a peripheral blood mononuclear cell isolated directly from a subject.

[0056] Aspect 11: The method of Aspect 10, wherein the regenerative potential is quantified by CD133 expression by the cell.

[0057] Aspect 12: The method of Aspect 10, wherein the regenerative potential is quantified by c-met expression by the cell.

[0058] Aspect 13: The method of Aspect 10, wherein the regenerative potential is quantified by CD34 expression by the cell.

[0059] Aspect 14: The method of Aspect 10, wherein the regenerative potential is quantified by IL-3 production when the cell is placed in 1% oxygen for at least 2 hours in vitro.

[0060] Aspect 15: The method of Aspect 1, wherein the polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second (i.e., a different) promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor.

[0061] Aspect 16: The method of Aspect 1, wherein the ligand is selected from the group comprising RG-115819, RG-115932, and RG-115830.

[0062] Aspect 17: The method of Aspect 1, wherein the ligand is an amidoketone or an oxadiazoline.

[0063] Aspect 18: The method of Aspect 1, wherein the ligand is administered less than one hour before or after the reprogrammed autologous cell.

[0064] Aspect 19: The method of Aspect 1, wherein the ligand is administered to the subject less than 1 hour, less than 24 hours, or less than 48 hours following administration of the reprogrammed autologous cell.IMMORTA-GMPRC-PCT

[0065] Aspect 20: The method of Aspect 1, wherein the reprogrammed autologous cell is a human cell.

[0066] Aspect 21: The method of Aspect 1, wherein the polynucleotide sequence encoding a gene switch comprises a polynucleotide sequence encoding a VP-16 transactivation domain.

[0067] Aspect 22: The method of Aspect 1, wherein the polynucleotide sequence encoding a gene switch comprises a polynucleotide sequence encoding a GAL-4 DNA binding domain.

[0068] Aspect 23: The method of Aspect 1, wherein the ecdysone receptor ligand binding domain comprises a substitution mutation.

[0069] Aspect 24: The method of Aspect 1, wherein the ecdysone receptor ligand binding domain is a Choristoneura fumiferana ecdysone receptor ligand binding domain.

[0070] Aspect 25: The method of Aspect 1, wherein the polynucleotide encoding a gene switch comprises a sequence encoding an RXR ligand binding domain.

[0071] Aspect 26: The method of Aspect 25, wherein the RXR binding domain is selected from the group comprising a vertebrate RXR ligand binding domain, an invertebrate RXR ligand binding domain, and a chimeric RXR ligand binding domain.

[0072] Aspect 27: The method of Aspect 25, wherein the RXR binding domain is a human RXR ligand binding domain.

[0073] Aspect 28: The method of Aspect 1, wherein the ligand is a diacylhydrazine.

[0074] Aspect 29: The method of Aspect 1, wherein the ligand is administered to the subject daily for a period of 14 days.

[0075] Aspect 30: The method of Aspect 1, wherein the subject is a human.

[0076] Aspect 31: The method of Aspect 1, wherein the reprogrammed autologous cell is detected in the subject 72 hours after administering the reprogrammed autologous cell to the subject and / or 72 hours after administering the ligand to the subject.IMMORTA-GMPRC-PCT

[0077] Aspect 32: The method of Aspect 1, wherein the ligand is administered to the subject orally.

[0078] Aspect 33: The method of Aspect 1, wherein the vector is an adenoviral vector.

[0079] Aspect 34: The method of Aspect 1, wherein the polypeptide encoding a gene switch

[0080] Aspect 35: The method of Aspect 1, wherein the polynucleotide encoding a gene switch comprises the following: (i) a first transcription factor sequence encoding a VP-16 transactivation domain and a chimeric RXR ligand binding domain; (ii) an EMCV IRES (i.e., internal ribosomal entry site from encephalomyocarditis virus); and (iii) a second transcription factor sequence encoding a GAL4 DNA-binding domain and a Choristoneura fumiferana ligand binding domain comprising a substitution mutation.

[0081] Aspect 36: The method of Aspect 35, wherein the ligand is RG-115932.

[0082] Aspect 37: The method of Aspect 35, wherein the ligand is a diacylhydrazine.

[0083] Aspect 38: The method of Aspect 37, wherein the diacylhydrazine is administered to the subject once daily for 14 consecutive days following administration of the reprogrammed autologous cell.

[0084] Aspect 39: The method of Aspect 1, wherein administering the in vitro reprogrammed autologous cell and the ligand induces an immune response in the subject.

[0085] Aspect 40: The method of Aspect 1, wherein administering the in vitro reprogrammed autologous cell and the ligand induces an immune response against a tumor in the subject.

[0086] Aspect 41: The method of Aspect 1, wherein the polynucleotide encoding a polypeptide sequence is selected based on a desired therapeutic effect on the reprogrammed autologous cell.IMMORTA-GMPRC-PCT

[0087] Aspect 42: The method of Aspect 1, wherein the polynucleotide encodes a molecule that is selected based on a desired therapeutic effect of the molecule on the reprogrammed autologous cell.

[0088] Aspect 43: The method of Aspect 1, wherein the reprogrammed autologous cell is transfected with a polynucleotide gene construct encoding an anti-apoptotic gene.

[0089] Aspect 44: The method of Aspect 43, wherein anti-apoptotic gene is selected from the group comprising obestatin, XIAP, sphingosine-1-phosphate, BCL-2, BCL-XL, GATA-4, IGF-1, EGF, heme-oxygenase-1, NF-kB, akt, pi3-k, and epha-2.

[0090] Aspect 45: The method of Aspect 43, wherein the in vivo persistence or survival of the reprogrammed autologous cell is endowed by modifying apoptosis of the cell.

[0091] Aspect 46: The method of Aspect 1, wherein the reprogrammed autologous cell is transfected with a polynucleotide gene construct capable of inducing RNA interference directed against a molecule associated with induction of apoptosis.

[0092] Aspect 47: The method of Aspect 46, wherein the molecule associated with induction of apoptosis is selected from the group comprising Fas, FasL, CASP1 (ICE), CASP10 (MCH4), CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR (CASPER), CRADD, PYCARD (TMS1 / ASC), ABL1, AKT1, BAD, BAK1, BAX, BCL2L11, BCLAF1, BID, BIK, BNIP3, BNIP3L, CASP1 (ICE), CASP10 (MCH4), CASP14, CASP2, CASP4, CASP6, CASP8, CD70 (TNFSF7), CIDEB, CRADD, FADD, FASLG (TNFSF6), HRK, LTA (TNFB), NOD1 (CARD4), PYCARD (TMS1 / ASC), RIPK2, TNF, TNFRSF10A, TNFRSF10B (DR5), TNFRSF25 (DR3), TNFRSF9, TNFSF10 (TRAIL), TNFSF8, TP53, TP53BP2, TRADD, TRAF2, TRAF3, and TRAF4.

[0093] Aspect 48: The method of Aspect 46, wherein in vivo persistence or survival of the reprogrammed autologous cell is endowed.

[0094] Aspect 49: The method of Aspect 1, wherein the reprogrammed autologous cell is administered intravenously, intrathecally, or intraventricularly.IMMORTA-GMPRC-PCT

[0095] Aspect 50: The method of Aspect 1, wherein the reprogrammed autologous cell is induced to differentiate into a neural cell lineage.

[0096] Aspect 51: The method of Aspect 50, wherein differentiation is induced by homing of the reprogrammed autologous cell to neural tissue following administration of the cell to the subject.

[0097] Aspect 52: The method of Aspect 50, wherein differentiation into the neural cell lineage is conferred by transfecting the cell with a polynucleotide gene construct encoding a polypeptide selected from the group comprising DCYAP1R1, ARTN, BDNF, CD40 (TNFRSF5), CNTF, CNTFR, CRHBP, CRHR1, CRHR2, FRS2, FRS3, FUS, GDNF, GFRA1, GFRA2, GFRA3, GMFB, GMFG, MAGED1, MT3, NF1, NGF, NGFR, NGFRAP1, NR1I2, NRG1, NRG2, NTF3, NTF4, NTRK1, NTRK2, PSPN, PTGER2, TFG, TRO, and VGF.

[0098] Aspect 53: The method of Aspect 50, wherein the reprogrammed autologous cell induces differentiation of endogenous neural progenitors in the subject.

[0099] Aspect 54: The method of Aspect 1, wherein the reprogrammed autologous cell is endowed with the ability to migrate or home to a tissue or a tumor site

[0100] Aspect 55: The method of Aspect 54, wherein migration or homing ability is conferred by transfecting the reprogrammed autologous cell with a polynucleotide gene construct encoding a polypeptide selected from the group comprising SDF-1, VEGF, FGF-1, FGF-2, PDGF-BB, angiopoietin, and TSPL-1.

[0101] Aspect 56: The method of Aspect 54, wherein the migration of a reprogrammed autologous cell comprises chemotaxis to an area of hypoxia.

[0102] Aspect 57: The method of Aspect 54, wherein the migration of a reprogrammed autologous cell comprises chemotaxis to an area of inflammation.

[0103] Aspect 58: The method of Aspect 54, wherein the migration of a reprogrammed autologous cell comprises chemotaxis to a tumor.

[0104] Aspect 59: The method of Aspect 1, wherein the reprogrammed autologous cell is endowed with the ability to stimulate angiogenesis.IMMORTA-GMPRC-PCT

[0105] Aspect 60: The method of Aspect 59, wherein the angiogenic ability is conferred by transfecting the reprogrammed autologous cell with a polynucleotide gene construct encoding a polypeptide selected from the group comprising activin A, adrenomedullin, aFGF, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4, bFGF, B61, bFGF inducing activity, cadherins, CAM-RF, cGMP analogs, ChDI, CLAF, claudins, collagen, collagen receptors .alpha..sub.1.beta..sub.1 and .alpha..sub.2.beta..sub.1, connexins, Cox-2, ECDGF (endothelial cell-derived growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, endothelins, endostatin, endothelial cell growth inhibitor, endothelial cell-viability maintaining factor, endothelial differentiation sphingolipid G-protein coupled receptor-1 (EDG1), ephrins, Epo, HGF, TGF-beta, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin and fibronectin receptor .alpha.5.beta.1, Factor X, HB-EGF, HBNF, HGF, HUAF, heart derived inhibitor of vascular cell proliferation, IL1, IGF-2 IFN-gamma, integrin receptors, K-FGF, LIF, leiomyoma-derived growth factor, MCP-1, macrophage-derived growth factor, monocyte-derived growth factor, MD-ECI, MECIF, MMP 2, MMP3, MMP9, urokinase plasminogen activator, neuropilin (NRP1, NRP2), neurothelin, nitric oxide donors, nitric oxide synthases (NOSs), notch, occludins, zona occludins, oncostatin M, PDGF, PDGF-B, PDGF receptors, PDGFR-.beta., PD- ECGF, PAI-2, PD-ECGF, PF4, P1GF, PKR1, PKR2, PPAR-gamma, PPAR-gamma ligands, phosphodiesterase, prolactin, prostacyclin, protein S, smooth muscle cell-derived growth factor, smooth muscle cell-derived migration factor, sphingosine-1-phosphate-1 (SIP1), Syk, SLP76, tachykinins, TGF-beta, Tie 1, Tie2, TGF-.beta., and TGF-.beta. receptors, TIMPs, TNF-alphatransferrin, thrombospondin, urokinase, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF.sub.164, VEGI, and EG-VEGF.

[0106] Aspect 61: The method of Aspect 1, wherein the reprogrammed autologous cell is endowed with the ability to stimulate an immune response in the subject such an anti-tumor response.

[0107] Aspect 62: The method of Aspect 61, wherein the immune stimulatory ability is conferred by transfecting the reprogrammed autologous cell with a polynucleotide gene construct encoding a polypeptide selected from the group comprising ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, CD40LGIMMORTA-GMPRC-PCT (TNFSF5), IFNA2, , IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL-6, IL8, IL9, IL-18, IL-33, LTA, LTB, MIF, SCYE1, SPP1, TNF, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), C5, CCL1 (I-309), CCL11 (eotaxin), HMGB1, IL-2. IL-12, IL-17, IL33. CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), complement component C3, complement component C5, 2,3 alpha gal, CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK) , CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC / IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9, IL13, and IL8.

[0108] Aspect 63: The method of Aspect 1, wherein the reprogrammed autologous cell is transfected with a polynucleotide gene construct encoding a polypeptide that is toxic to cancer cells.

[0109] Aspect 64: The method of Aspect 63, wherein the polypeptide that is toxic to cancer cells comprises TRAIL, TNF-alpha, IL-12, IL-15, IFN-gamma, and IFN-alpha.

[0110] Aspect 65: The method of Aspect 1, wherein the reprogrammed autologous cell is a progenitor cell.

[0111] Aspect 66: The method of Aspect 65, wherein the progenitor cell is derived from a human induced pluripotent stem cell.

[0112] Aspect 67: The method of Aspect 65, wherein the progenitor cell expresses one or a plurality of markers comprising CD29, CD41a, CD44, CD90, and CD105.

[0113] Aspect 68: The method of Aspect 1, wherein the reprogrammed autologous cell expresses one or a plurality of markers comprising NeuN, CD9, CD62, CD59, Actin, GFAP, NSE, Nestin, CD73, SSEA-4, hTERT, Oct-4, and tubulin.

[0114] Aspect 69: The method of Aspect 1, wherein the reprogrammed autologous cell proliferates at a rate of 0.5-1.5 doublings per 24 hours in a growth medium.

[0115] Aspect 70: The method of Aspect 1, wherein the reprogrammed autologous cell expresses a marker selected from hTERT and Oct-4, or both.IMMORTA-GMPRC-PCT

[0116] Aspect 71: The method of Aspect 70, wherein the reprogrammed autologous cell does not express STRO-1.

[0117] Aspect 72: The method of Aspect 70, wherein the reprogrammed autologous cell undergoes cell division within 24 hours of culture in a growth medium.

[0118] Aspect 73: The method of Aspect 1, wherein the reprogrammed autologous cell expresses STRO-1.

[0119] Aspect 74: The method of Aspect 73, wherein the reprogrammed autologous cell proliferates at the rate of 0.5-0.9 doublings per 24 hours in a growth medium.

[0120] Aspect 75: The method of Aspect 1, wherein the reprogrammed autologous cell produces molecules selected from the group comprising matrix metalloprotease 3 (MMP3), matrix metalloprotease 10 (MMP10), granulocyte-macrophage colony- stimulating factor (GM-CSF), platelet derived growth factor BB (PDGF-BB), and angiopoietin-2 (ANG-2.

[0121] Aspect 76: The method of Aspect 1, wherein the reprogrammed autologous cell originates or is derived from a pluripotent cell.

[0122] Aspect 77: The method of Aspect 1, wherein the reprogrammed autologous cell is capable of differentiating into one or a plurality of cell types or lineages comprising an adipogenic, endothelial, hepatic, osteogenic, neural, pancreatic or myocytic cell lineage.

[0123] Aspect 78: The method of Aspect 1, wherein the reprogrammed autologous cell has a stable karyotype for at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more cell divisions.

[0124] In one embodiment, the invention teaches addition of anti-apoptotic molecules to mesenchymal stem cells generated from pluripotent stem cells, which may enhance therapeutic efficacy by increasing in vivo lifespan. The invention teaches the transfection of genes such as XIAP, BCL-2, BCL-Xl, or survivin, increased potency of cell therapy. Such genes may be considered. Conversely, shRNA may be utilized to silence proapoptotic molecules such as BAX or BCL-Xs.IMMORTA-GMPRC-PCT

[0125] One possible area of modulating efficacy is through transfecting MSC with various homing molecules. In one specific possibility, cells may be transfected with CXCR4 to further augment homing to areas of ischemia. Additional adhesion molecules may be considered that are associated with specific checkpoints of cellular extravasation and targeting to specific organs or tissues.

[0126] Production of neurotrophic growth factors by MSC through transfection has been contemplated by the inventors. Candidate factors such as NGF, NT, CNGF, and others have been considered with the concept of enhancing neurotrophic activity in conditions such as stroke, where the combination of angiogenesis and neurogenesis has been described as essentially for therapeutic response.

[0127] Other areas of transfection with growth factors include the space of musculoskeletal defects including non-union bone fracture, where BMP2 or 4 would be considered for transfection.

[0128] Another direction would be the transfection of MSC with SDF-1 in order to allow MSC to recruit other endogenous stem cell types in order to repair injured or ischemic tissues.

[0129] MSC may be transfected with shRNA targeting Th17 or Th1 cytokines in order to prevent in vivo immune deviation of the MSC into an inflammatory cell type. Although this has not been observed to date with MSC. Alternatively, cells may be transfected with “master” immune modulatory transcription factors to induce Th2 or Tr1 cytokine production from the cells. Molecules of interest include STAT6. Other immune modulatory molecules of interest include IL-10, IL-35 (for Treg generation), and PD1L

[0130] The ability of the MSC to home to hypoxic environments may allow for use of the cell as a vector to target tumor stem cells. MSC may be transfected with toxic genes such as TRAIL, especially if an inducible system for gene expression was available. This is interesting in light of published data showing non-transfected MSC exerted anticancer effects in a small animal glioma model.

[0131] Another cancer-targeting approach, especially if an inducible vector was found, would be to transfect MSC with the gene encoding alpha1,3-galactosyltransferase (alpha-GT), this would allow for cell-surface generation of alpha-Gal, one of the mostIMMORTA-GMPRC-PCT immunogenic peptides that causes complement activation. The concept would be to administer transfected MSC, allow for homing to tumor, then induce gene expression locally, which would cause significant tumor damage via complement activation.

[0132] Cord blood transplantation has been used successfully in some mucopolysaccharide disorders such as Hurlers Syndrome. Given that MSC disseminate systemically, successful transfection of some of the MPS enzymes may allow for targeting of this Orphan Drug Space which companies like BioMarin have been actively engaged in using enzyme replacement therapy. Some of the therapeutic activities of MSC occur via exosomes released from these cells. One possible approach would be to use exosome targeting sequences to generate MSC whose exosomes are either specifically targeted to a certain tissue or disease pathology, or to utilize the exosomes a drugs.

[0133] In some embodiments, transfecting MSC with differentiation inducing genes can be utilized to generate insulin producing cells.

[0134] The recent discovery that GnRH decline is associated with aging, and local administration of this hormone reverses signs of aging, suggests the possibility of utilizing GnRH transfected MSC as a mechanism of possibly slowing aging, or aging associated pathology.

[0135] In one embodiment of the invention, an EcR complex can be activated by an active ecdysteroid or non-steroidal ligand bound to one of the proteins of the complex, inclusive of EcR, but not excluding other proteins of the complex. As used herein, the term "ligand," as applied to EcR-based gene switches, describes small and soluble molecules having the capability of activating a gene switch to stimulate expression of a polypeptide encoded therein. Examples of ligands include, without limitation, an ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazines such as those disclosed in U.S. Pat. Nos.6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S. Published Application Nos.2005 / 0209283 and 2006 / 0020146; oxadiazolines as described in U.S. Published Application No.2004 / 0171651; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No.461,809; N-alkyl- N,N'-diaroylhydrazines such as those disclosed in U.S. Pat. No.5,225,443; N-acyl-N- alkylcarbonylhydrazines such as those disclosed in European Application No.234,994;IMMORTA-GMPRC-PCT N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U.S. Pat. No.4,985,461; amidoketones such as those described in U.S. Published Application No.2004 / 0049037; and other similar materials including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7- ketocholesterol-3-sulfate, famesol, bile acids, 1,1-biphosphonate esters, juvenile hormone III, and the like. Examples of diacylhydrazine ligands useful in the invention include RG- 115819 (3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-methyl-3- methoxy-benzoyl)-hydrazide- ), RG-115932 ((R)-3,5-Dimethyl-benzoic acid N-(1-tert- butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-115830 (3,5-Dimethyl- benzoic acid N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide). See U.S. application Ser. No.12 / 155,111, filed May 29, 2008, and PCT / US2008 / 006757 filed May 29, 2008, for additional diacylhydrazines that are useful in the practice of the invention. The EcR complex includes proteins which are members of the nuclear receptor superfamily wherein all members are characterized by the presence of an amino- terminal transactivation domain ("TA"), a DNA binding domain ("DBD"), and a ligand binding domain ("LBD") separated by a hinge region. Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD. The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for ecdysone response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins.

[0136] DNA sequences making up the exogenous gene, the response element, and the EcR complex may be incorporated into archaebacteria, procaryotic cells such as Escherichia coli, Bacillus subtilis, or other enterobacteria, or eucaryotic cells such as plant or animal cells. However, because many of the proteins expressed by the gene are processed incorrectly in bacteria, eucaryotic cells are preferred. The cells may be in the form of single cells or multicellular organisms. The nucleotide sequences for the exogenous gene, the response element, and the receptor complex can also be incorporated as RNA molecules, preferably in the form of functional viral RNAs such as tobacco mosaic virus. Of the eucaryotic cells, vertebrate cells are preferred because they naturally lack the molecules which confer responses to the ligands of this invention for the EcR. As a result, they are "substantially insensitive" to the ligands of this invention. Thus, theIMMORTA-GMPRC-PCT ligands useful in this invention will have negligible physiological or other effects on transformed cells, or the whole organism. Therefore, cells can grow and express the desired product, substantially unaffected by the presence of the ligand itself. In certain embodiments of the invention EcR ligands, when used with the EcR complex which in turn is bound to the response element linked to an exogenous gene (e.g., IL-12), provide the means for external temporal regulation of expression of the exogenous gene. The order in which the various components bind to each other, that is, ligand to receptor complex and receptor complex to response element, is not critical. Typically, modulation of expression of the exogenous gene is in response to the binding of the EcR complex to a specific control, or regulatory, DNA element. The EcR protein, like other members of the nuclear receptor family, possesses at least three domains, a transactivation domain, a DNA binding domain, and a ligand binding domain. This receptor, like a subset of the nuclear receptor family, also possesses less well-defined regions responsible for heterodimerization properties. Binding of the ligand to the ligand binding domain of EcR protein, after heterodimerization with USP or RXR protein, enables the DNA binding domains of the heterodimeric proteins to bind to the response element in an activated form, thus resulting in expression or suppression of the exogenous gene. This mechanism does not exclude the potential for ligand binding to either EcR or USP, and the resulting formation of active homodimer complexes (e.g., EcR+EcR or USP+USP). In one embodiment, one or more of the receptor domains can be varied producing a chimeric gene switch. Typically, one or more of the three domains may be chosen from a source different than the source of the other domains so that the chimeric receptor is optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988) or LexA protein from E. coli (see Brent et al., Cell 43:729 (1985)) to accommodate chimeric EcR complexes. Another advantage of chimeric systems is that they allow choice of a promoter used to drive the exogenous gene according to a desired end result. Such double control can be particularly important in areas of gene therapy, especially when cytotoxic proteins are produced, because both the timing of expression as well as the cells wherein expression occurs can be controlled. When exogenous genes, operatively linked to a suitable promoter, are introduced into the cells of the subject, expression of the exogenous genes is controlled by the presence of the ligand of thisIMMORTA-GMPRC-PCT invention. Promoters may be constitutively or inducibly regulated or may be tissue- specific (that is, expressed only in a particular type of cell) or specific to certain developmental stages of the organism.

[0137] For the purpose of the invention, the gene switch may be any gene switch system that regulates gene expression by addition or removal of a specific ligand. In one embodiment, the gene switch is one in which the level of gene expression is dependent on the level of ligand that is present. Examples of ligand-dependent transcription factors that may be used in the gene switches of the invention include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect of the invention, the gene switch is an EcR-based gene switch. Examples of such systems include, without limitation, the systems described in U.S. Pat. Nos.6,258,603, 7,045,315, U.S. Published Patent Application Nos.2006 / 0014711, 2007 / 0161086, and International Published Application No. WO 01 / 70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No.7,091,038, U.S. Published Patent Application Nos.2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457, and 2006 / 0100416, and International Published Application Nos. WO 01 / 70816, WO 02 / 066612, WO 02 / 066613, WO 02 / 066614, WO 02 / 066615, WO 02 / 29075, and WO 2005 / 108617. An example of a non- steroidal ecdysone agonist-regulated system is the RheoSwitch.RTM. Mammalian Inducible Expression System (New England Biolabs, Ipswich, Mass.).

[0138] In one specific embodiment, a polynucleotide encoding the gene switch comprises a single transcription factor sequence encoding a ligand-dependent transcription factor under the control of a promoter. The transcription factor sequence may encode a ligand-dependent transcription factor that is a naturally occurring or an artificial transcription factor. An artificial transcription factor is one in which the natural sequence of the transcription factor has been altered, e.g., by mutation of the sequence or by the combining of domains from different transcription factors. In one embodiment, the transcription factor comprises a Group H nuclear receptor ligand binding domain (LBD). In one embodiment, the Group H nuclear receptor LBD is from an EcR, a ubiquitous receptor, an orphan receptor 1, a NER-1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor.beta., a steroid hormone receptor likeIMMORTA-GMPRC-PCT protein, a liver X receptor, a liver X receptor.alpha., a farnesoid X receptor, a receptor interacting protein 14, or a farnesol receptor. In another embodiment, the Group H nuclear receptor LBD is from an ecdysone receptor. The EcR and the other Group H nuclear receptors are members of the nuclear receptor superfamily wherein all members are generally characterized by the presence of an amino-terminal transactivation domain (TD), a DNA binding domain (DBD), and a LBD separated from the DBD by a hinge region. As used herein, the term "DNA binding domain" comprises a minimal polypeptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response element. Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: A / B, C, D, E, and in some members F (see U.S. Pat. No.4,981,784 and Evans, Science 240:889 (1988)). The "A / B" domain corresponds to the transactivation domain, "C" corresponds to the DNA binding domain, "D" corresponds to the hinge region, and "E" corresponds to the ligand binding domain. Some members of the family may also have another transactivation domain on the carboxy- terminal side of the LBD corresponding to "F". The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins. The EcR, like a subset of the nuclear receptor family, also possesses less well-defined regions responsible for heterodimerization properties. Because the domains of nuclear receptors are modular in nature, the LBD, DBD, and TD may be interchanged.

[0139] In another embodiment, the transcription factor comprises a TD, a DBD that recognizes a response element associated with the exogenous gene whose expression is to be modulated, and a Group H nuclear receptor LBD. In certain embodiments, the Group H nuclear receptor LBD comprises a substitution mutation. In other embodiments, a polynucleotide encoding the gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor, i.e., a "dual switch"- or "two-hybrid"-based gene switch. The first and second promoters may be the same or different. A polynucleotide encoding a gene switch may also comprise a firstIMMORTA-GMPRC-PCT transcription factor sequence and a second transcription factor sequence under the control of one promoter, wherein the proteins encoded by the first transcription factor sequence and the second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor, i.e., a "single gene switch." The first transcription factor sequence and the second transcription factor sequence may be connected by an internal ribosomal entry site, e.g., EMCV IRES. In one embodiment, the first transcription factor sequence encodes a polypeptide comprising a TD, a DBD that recognizes a response element associated with the exogenous gene whose expression is to be modulated; and a Group H nuclear receptor LBD, and the second transcription factor sequence encodes a transcription factor comprising a nuclear receptor LBD selected from a vertebrate RXR LBD, an invertebrate RXR LBD, an ultraspiracle protein LBD, and a chimeric LBD comprising two polypeptide fragments, wherein the first polypeptide fragment is from a vertebrate RXR LBD, an invertebrate RXR LBD, or an ultraspiracle protein LBD, and the second polypeptide fragment is from a different vertebrate RXR LBD, invertebrate RXR LBD, or ultraspiracle protein LBD. In another embodiment, the gene switch comprises a first transcription factor sequence encoding a first polypeptide comprising a nuclear receptor LBD and a DBD that recognizes a response element associated with the exogenous gene whose expression is to be modulated, and a second transcription factor sequence encoding a second polypeptide comprising a TD and a nuclear receptor LBD, wherein one of the nuclear receptor LBDs is a Group H nuclear receptor LBD. In a preferred embodiment, the first polypeptide is substantially free of a TD and the second polypeptide is substantially free of a DBD. For purposes of the invention, "substantially free" means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.

[0140] For production of cells, reagent qualification may be necessary. The qualification process begins with the vender of the reagent. The vender is qualified through our standard operating procedure. A corresponding form is completed and approval gained before a vender can be used. The Criteria identified as important in qualifying a supplier include quality of product, services offered, competitive pricing, communication, availability, how complaints are handled and the overall fit to our systems. This list is not all inclusive. Quality Systems reviews each qualification form and will approve based on the criteria stated above. Once the vender is approved, they are added to the Supplies and Services List. Associates ordering supplies includingIMMORTA-GMPRC-PCT reagents use the list. Only approved venders on the list are used by associates ordering supplies involving reagents. Once the reagent arrives, it is logged on the Supplies Receipt, Inspection and Inventory Log. The form instructs the associate to complete certain information for the incoming reagent. These fields are date received, initials of receiver, name of the item, manufacturer, lot number, expiration date, package passed visual inspection, product passed visual inspection, date available for use and quantity. The COA is examined for reagents and placed in the applicable COA binder under that reagent name. These binders are retained per the record retention procedure. Once this is completed the reagent is released from quarantine and placed in the applicable area. If the reagent needs refrigerated or is to remain frozen, it is placed in the applicable storage environment. FDA or other national regulatory body-approved reagents are used if available. In one embodiment, an excipient used in the cryopreservation of the cells is Dimethyl Sulfoxide (DMSO). Each dose of MSC is cryopreserved using 10% DMSO, or 2 mL of DMSO in a total volume of 10 mL of final product. Infusion of this amount of DMSO is well within the safety parameters for a 30 kg child; Pediatric Stem Cell Transplant SOP states that the maximum dose of DMSO is 15 mg / kg / dose.

[0141] During the process of manufacturing, it is ideal for the production to occur in a class 10,000 clean production suite. Each technician properly gowns when entering in the GMP room. Before entry into the clean lab area, the technician obtains a bunny suit in the ante room. After the hood of the bunny suit is placed on, a mouth covering is put on, making sure that all hair is fully covered under the hood and mouth covering. The technician puts on a pair of sterile powder free gloves, and entera the clean lab space with the sample. Environmental monitoring is performed in the Class 10,000 clean room. The clean room uses Biological Safety Cabinets (BSC) which maintains a Class 5 environment. BSC are certified annually by an outside qualified vender. Settling plates are performed every time the BSC is in use for processing and evaluated for acceptable criteria based on USP. One settling plate is placed in the BSC during processing for a minimum of 30 minutes. Once per package, as a negative control, one covered settling plate is placed inside the BCS at the same time. After the settling plate is in the BSC, evaluate the plate for presence of bacterial colonies, Colony Forming Units (cfu), by allowing the plate to incubate for 48 hours at 37ºC. Levels requiring alert are more than 1 colony per plate. Incubator temperature should be 36-38oC. TSA plates are used to evaluate the wide spectrum of possible bacteria present. Prepared plates are in theirIMMORTA-GMPRC-PCT original wrapping at 2 – 8ºC and are warmed to room temperature prior to use. The product is validated from the time of manufacture to be stable at room temperature (25ºC) for 192 h (8 days). Additionally, the clean room is monitored for room temperature and particle counts. Acceptable room temperature is between 15 and 30 degrees Celsius. A MetOne Aerocet 531 particle counter, or alternative, may be used to evaluate the particles in the air. The particle counter is used to detect and count the number of particles found in the air of the clean room. It is used to confirm that the number of loose particles in the air is less than 10,0000.5 micron particles per ft3. The particle counter is run on a weekly basis in the three major areas of the clean room space. It is run for 30 minutes each in the gowning area, on the counter inside the clean room space and inside the hood. A settle plate is placed each time the particle counter is in use, next to the counter for the 30 minutes it is being run. After each use of the clean room, the BSC is wiped down with 5.25% bleach then followed by a 70% isopropyl alcohol. Countertops inside the clean room space are wiped down with 70% isopropyl alcohol each day. Once a week all surfaces inside the clean room, including floor, are wiped down with enzymatic cleaner LpH using a dry disposable cloth. Yearly, all walls and ceiling are clean with a lint roller, and all soft walls are cleaned with 70% isopropyl alcohol. Before laboratory technicians are allowed into the clean room, a gowning competency must be passed. RODAC plates are utilized to assess the competency of the technician. The acceptable limits of CFU / plate are determined according to local regulations. In one example, the following limits are used: Finger tips 10, CFU / plate, Gown Zipper 5 CFU / plate, Gown Lower Sleeve Area 5 CFU / plate, Hood Corner 5 CFU / Plate, Floor Surface 10 CFU / plate. Genome editing using programmable nucleases enables precise editing at specific genomic loci, which can be used to remove deleterious mutations or insert protective mutations. To date, there are three major classes of nucleases—zinc finger nucleases (ZFNs), transcription activator- like effector nucleases (TALENs), and clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated nucleases. Of these, CRISPR-associated nucleases have proven to be markedly superior to the others in terms of the ease and simplicity of use.

[0142] The CRISPR / Cas system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a ‘seed’ sequence within the guide RNA (gRNA) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The Cas9 protein, under direction from the gRNA, binds to its target DNA sequence and cutsIMMORTA-GMPRC-PCT both strands of the DNA at a specific locus. This double-stranded DNA break is repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ frequently causes small insertions or deletions (indels) at the breakage site that can lead to a frameshift mutation of the protein encoded by the gene. HDR utilizes a repair template that is copied into the gene, thus engineering specific mutations.

[0143] The CRISPR / CAS system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, CAR T cells, and stem and progenitor cells. In one aspect, the invention includes a modified hematopoietic stem or progenitor cell comprising a nucleic acid capable of decreasing expression of an endogenous gene or a portion thereof, wherein the endogenous gene encodes a polypeptide comprising an antigen domain targeted by a chimeric antigen receptor (CAR).

[0144] One example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Publication No.2014 / 0068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.

[0145] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. The CRISPR / CAS system can also simultaneously target multiple genomic loci by co-expressing a single CAS9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergistic activation of target genes. In one aspect, a modified hematopoietic stem or progenitor cell is generated by introducing a nucleic acid capable of decreasing expression of an endogenous gene or a portion thereof into the cell, wherein the endogenous gene encodes a polypeptide comprising an antigen domain targeted by a chimeric antigen receptor (CAR). In such an embodiment, the nucleic acid capable of decreasing expression of the endogenous gene or a portion thereof is a CRISPR system. In some embodiments, the CRISPR systemIMMORTA-GMPRC-PCT includes a Cas expression vector and a guide nucleic acid sequence specific for the endogenous gene. In another embodiment, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, other nucleases known in the art, and any combination thereof.

[0146] In one embodiment, introducing the CRISPR system comprises introducing an inducible CRISPR system. The CRISPR system may be induced by exposing the hematopoietic stem or progenitor cell to an agent that activates an inducible promoter in the CRISPR system, such as the Cas expression vector. In such an embodiment, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline). However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

[0147] The guide nucleic acid sequence is specific for a gene and targets that gene for Cas endonuclease-induced double strand breaks. The sequence of the guide nucleic acid sequence may be within a locus of the gene. In one embodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

[0148] Methods of introducing a nucleic acid into the hematopoietic stem or progenitor cell include physical, biological and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, mircoinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany). RNA can also be introduced into cells using cationic liposome mediated transfection using lipofection using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).IMMORTA-GMPRC-PCT

[0149] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

[0150] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0151] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about −20° C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.IMMORTA-GMPRC-PCT

[0152] Regardless of the method used to introduce the nucleic acid into the cell, a variety of assays may be performed to confirm the presence of the nucleic acid in the cell. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0153] In one aspect, the invention includes a method for generating a modified hematopoietic stem or progenitor cell comprising introducing a nucleic acid capable of decreasing endogenous gene expression into the cell, wherein the endogenous gene encodes a polypeptide comprising an antigen domain to be targeted by a chimeric antigen receptor (CAR). In one embodiment, the method further comprises introducing a modified endogenous gene into the modified cell, wherein the modified endogenous gene encodes a modified polypeptide lacking the antigen domain targeted by the CAR. In such an embodiment, one nucleic acid may be introduced using the same or a different method from that used to introduce the modified endogenous gene into the cell. The guide nucleic acid sequence may be specific for any gene, such as an endogenous gene that would reduce immunogenicity or reduce sensitivity to a CART therapy. The endogenous gene of the present invention encodes a polypeptide comprising an antigen domain targeted by a CAR. In one embodiment, the guide nucleic acid sequence is specific for the endogenous gene that encodes a tumor antigen. In yet another embodiment, the guide nucleic acid sequence is specific for the endogenous gene that encodes CD33 or CD123.

[0154] The guide nucleic acid sequence includes an RNA sequence, a DNA sequence, a combination thereof (an RNA-DNA combination sequence), or a sequence with synthetic nucleotides. The guide nucleic acid sequence can be a single molecule or a double molecule. In one embodiment, the guide nucleic acid sequence comprises a single guide RNA.

[0155] Chimeric antigen receptors (CARs) are typically used as a therapy in adoptive cell transfer. The CAR is an artificial receptor expressed on a T cell (i.e., a CAR T cell) that is engineered to specifically bind to an antigen and activate the T cell as an immune effector cell. In many instances, the antigen targeted by the CART cells is anIMMORTA-GMPRC-PCT endogenous gene that is expressed on normal and diseased cells. Thus, the CART cells target both normal and diseased cells for elimination.

[0156] The target of the CAR of the present invention encodes an endogenous (to the cell) polypeptide comprising an antigen domain expressed on cells. A CAR usually includes an extracellular domain that comprises an antigen binding domain. In some embodiments, the antigen binding domain of the CAR specifically binds to the antigen on a target cell. In other embodiments, the antigen binding domain of the CAR specifically binds to a tumor antigen. In one embodiment, the endogenous gene is expressed on a tumor cell targeted by the CAR. In some embodiments, the endogenous gene encodes a cell surface molecule comprising an antigen domain targeted by the CAR. Cell surface molecules include endogenous molecules that may act as a binding partner associated with viral, bacterial and parasitic infections.

[0157] Examples of endogenous genes may include, but are not limited to a gene that encodes CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (CD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlep(1-1)Cer); TNF receptor family member B cell maturation (BCMA). To antigen ((Tn Ag) or (GalNAcα-Ser / Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like tyrosine Kinase 3 (FLT3); Tumor- associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2 / neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor I receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type 9IMMORTA-GMPRC-PCT (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr- abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex; locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer / testis antigen 1 (NY-ESO- 1); Cancer / testis antigen 2 (LAGE-1a); Melanoma-associated antigen 1 (MAGE-A1), ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-1AP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P4501B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, XIMMORTA-GMPRC-PCT breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxy esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0158] The choice of endogenous gene depends on the expression of the endogenous gene in normal cells, the presence of the expressed endogenous gene product on the surface of normal cells, and the effect CART therapy has on normal cells. For example, the endogenous gene may be highly expressed on diseased cells and have limited expression in normal cells.

[0159] In another example, the endogenous gene may be widely expressed in normal blood cells and the CART therapy would potentially target all those cells in addition to diseased cells. In such circumstances, introduction of a modified endogenous gene may be useful. Introduction of a modified endogenous gene may be particularly useful when expression of the endogenous gene is critical for the health of a non-diseased cell, such as a hematopoietic stem or progenitor cell. In one embodiment, the method of generating the modified cell described herein comprises decreasing expression of a portion of the endogenous gene, such as the portion comprising the antigen domain targeted by the CAR. In such an embodiment, the method can comprise introducing a modified endogenous gene that encodes a modified polypeptide lacking the antigen domain targeted by the CAR. In another embodiment, the modified cell described herein comprises a modified endogenous gene that encodes a modified polypeptide lacking the antigen domain targeted by the CAR. In yet another embodiment, the modified polypeptide comprises at least one function that is equivalent to the function of the polypeptide encoded by the endogenous gene. Thus, the modified cell expresses the modified endogenous gene, while maintaining resistance to CART cell therapy. The cellIMMORTA-GMPRC-PCT type generated according to the above description is characterized by expression of unique molecules. This is in contrast to other types of stem cells. The most commercially developed stem cell type outside of the hematopoietic sphere is the bone marrow derived mesenchymal stem cell. This cell population expresses significantly higher concentrations of the following genes as compared to bone marrow mesenchymal cells: somatostatin receptor 1, forkhead box L2, FAM105A, synaptopodin 2-like, anoctamin 4, spondin 2, CARD16, VAT1L, indolethylamine N-methyltransferase, deiodinase, iodothyronine, type II (DIO2), aldehyde dehydrogenase 1 family, member A1, 5-hydroxytryptamine (serotonin) receptor 2B, caspase recruitment domain family, member 17, Rho GTPase activating protein 20, zinc finger and BTB domain containing 46 (ZBTB46), synaptopodin 2-like (SYNPO2L), transcript variant 1, mRNA, chromosome 13 open reading frame 15 (C13orf15), mRNA, homeobox D11 (HOXD11), mRNA, oxidized low density lipoprotein (lectin-like) receptor 1 (OLR1), transcript variant 2, mRNA, homeobox D10 (HOXD10), mRNA, Pregnancy specific beta-1- glycoprotein 4, matrix metallopeptidase 3 (stromelysin 1, progelatinase) (MMP3), mRNA, actin filament associated protein 1-like 2 (AFAP1L2), transcript variant 2, mRNA,SH3-domain GRB2-like (endophilin) interacting protein 1 (SGIP1), mRNA, solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 (SLC7A2), transcript variant 1, mRNA, pregnancy specific beta-1-glycoprotein 8 (PSG8), transcript variant 3, mRNA, Wilms tumor 1 (WT1), transcript variant F, mRNA, platelet-derived growth factor beta polypeptide (PDGFB), transcript variant 1, mRNA, forkhead box F1 (FOXF1), mRNA, mannan-binding lectin serine peptidase 1 (C4 / C2 activating component of Ra-reactive factor) (MASP1), transcript variant 1, mRNA, deiodinase, iodothyronine, type II (DIO2), transcript variant 4, mRNA, G protein-coupled receptor 126 (GPR126), transcript variant b1, mRNA, stimulated by retinoic acid gene 6 homolog (mouse) (STRA6), transcript variant 6, mRNA, hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), transcript variant 2, mRNA, RAS guanyl releasing protein 2 (calcium and DAG-regulated) (RASGRP2), transcript variant 4, mRNA, membrane bound O- acyltransferase domain containing 1 (MBOAT1), mRNA, LON peptidase N-terminal domain and ring finger 2 (LONRF2), mRNA, interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), mRNA, cathepsin C (CTSC), transcript variant 3, mRNA, caspase 1, apoptosis-related cysteine peptidase (interleukin 1, beta, convertase) (CASP1), transcript variant beta, mRNA, integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), mRNA, integrin, alpha 6 (ITGA6), transcriptIMMORTA-GMPRC-PCT variant 1, mRNA, contactin 3 (plasmacytoma associated) (CNTN3), mRNA, synaptophysin-like 2 (SYPL2), mRNA, Rho GTPase activating protein 25 (ARHGAP25), transcript variant 4, mRNA, potassium voltage-gated channel, Shal-related subfamily, member 2 (KCND2), mRNA, S100 calcium binding protein A4 (S100A4), transcript variant 1, mRNA, chemokine (C-C motif) receptor-like 2 (CCRL2), transcript variant 2, mRNA, integrin, alpha 6 (ITGA6), transcript variant 2, mRNA, guanylate binding protein 4 (GBP4), mRNA, multiple C2 domains, transmembrane 1 (MCTP1), transcript variant S, mRNA, interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), mRNA, C1q and tumor necrosis factor related protein 9 (C1QTNF9), mRNA, progesterone receptor (PGR), transcript variant 2, mRNA, hypothetical LOC646113 (FLJ43390), non-coding RNA. zinc finger and BTB domain containing 46 (ZBTB46), mRNA, interferon-induced protein with tetratricopeptide repeats 3 (IFIT3), transcript variant 1, mRNA, microphthalmia-associated transcription factor (MITF), transcript variant 2, mRNA, interleukin 24 (IL24), transcript variant 4, mRNA, neuronal pentraxin I (NPTX1), mRNA, integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), mRNA, WT1 antisense RNA (non-protein coding) (WT1-AS), non-coding RNA, microphthalmia-associated transcription factor (MITF), transcript variant 2, mRNA, sterile alpha motif domain containing 12 (SAMD12), transcript variant 1, mRNA, chromosome 7 open reading frame 58 (C7orf58), transcript variant 1, mRNA, phospholipase B domain containing 1 (PLBD1), mRNA, podocalyxin-like (PODXL), transcript variant 2, mRNA, interleukin 8 (IL8), mRNA, chromosome 7 open reading frame 58 (C7orf58), transcript variant 2, mRNA, protein kinase, cAMP-dependent, regulatory, type II, beta (PRKAR2B), mRNA, chromosome 8 open reading frame 4 (C8orf4), mRNA, stimulated by retinoic acid gene 6 homolog (mouse) (STRA6), transcript variant 5, mRNA, progesterone receptor (PGR), transcript variant 2, mRNA, angiopoietin 2 (ANGPT2), transcript variant 2, mRNA, synaptophysin-like 2 (SYPL2), mRNA, retinoic acid receptor responder (tazarotene induced) 2 (RARRES2), mRNA, claudin 1 (CLDN1), mRNA, sal-like 1 (Drosophila) (SALL1), transcript variant 1, mRNA, calcium channel, voltage-dependent, T type, alpha 1H subunit (CACNA1H), transcript variant 2, mRNA, armadillo repeat containing 4 (ARMC4), mRNA, phosphatidylinositol-specific phospholipase C, X domain containing 3 (PLCXD3), mRNA, G protein-coupled receptor, family C, group 5, member A (GPRC5A), mRNA, GATA binding protein 2 (GATA2), transcript variant 1, mRNA, actin filament associated protein 1-like 1 (AFAP1L1), transcript variant 1, mRNA, ST6 (alpha-N-acetyl-IMMORTA-GMPRC-PCT neuraminyl-2,3-beta-galactosyl-1, 3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 (ST6GALNAC5), mRNA, H19, imprinted maternally expressed transcript (non-protein coding) (H19), non-coding RNA, sphingosine-1-phosphate receptor 3 (S1PR3), mRNA, protein kinase, cAMP-dependent, regulatory, type II, beta (PRKAR2B), mRNA, interleukin 7 receptor (IL7R), mRNA, calcium / calmodulin-dependent protein kinase IG (CAMK1G), mRNA, homeobox A11 (HOXA11), mRNA, renin (REN), mRNA, transcription factor AP-2 gamma (activating enhancer binding protein 2 gamma) (TFAP2C), mRNA, cathepsin C (CTSC), transcript variant 2, mRNA, STAM binding protein-like 1 (STAMBPL1), mRNA, Hypothetical protein LOC254057, adrenomedullin (ADM), mRNA, tenascin XB (TNXB), transcript variant XB-S, mRNA, KH domain containing, RNA binding, signal transduction associated 3 (KHDRBS3), mRNA, solute carrier family 35, member F3 (SLC35F3), mRNA, collagen, type IV, alpha 6 (COL4A6), transcript variant B, mRNA, protocadherin 7 (PCDH7), transcript variant a, mRNA, chromosome 10 open reading frame 58 (C10orf58), transcript variant 1, mRNA, fibroblast growth factor 9 (glia-activating factor) (FGF9), mRNA, sushi domain containing 3 (SUSD3), mRNA, melanoma associated antigen (mutated) 1-like 1 (MUM1L1), transcript variant 2, mRNA, keratin 18 pseudogene (FLJ40504), non-coding RNA, C-type lectin domain family 14, member A (CLEC14A), mRNA, 1-acylglycerol-3- phosphate O-acyltransferase 9 (AGPAT9), mRNA, phosphodiesterase 8B (PDE8B), transcript variant 3, mRNA, ArfGAP with dual PH domains 2 (ADAP2), mRNA, Keratin 18, family with sequence similarity 65, member C (FAM65C), mRNA, androgen receptor (AR), transcript variant 1, mRNA, phosphodiesterase 9A (PDE9A), transcript variant 2, mRNA, Intercellular adhesion molecule 1, monoglyceride lipase (MGLL), transcript variant 2, mRNA, HOXA11 antisense RNA 1 (non-protein coding) (HOXA11-AS1), antisense RNA, receptor (chemosensory) transporter protein 4 (RTP4), mRNA, reticulon 4 receptor (RTN4R), mRNA, Keratin pseudogene, annexin A3 (ANXA3), mRNA, RAS guanyl releasing protein 2 (calcium and DAG-regulated) (RASGRP2), transcript variant 3, mRNA, serum deprivation response (SDPR), mRNA, collagen, type XIII, alpha 1 (COL13A1), transcript variant 21, mRNA, v-ets erythroblastosis virus E26 oncogene homolog 2 (avian) (ETS2), mRNA, ADAM metallopeptidase domain 8 (ADAM8), transcript variant 3, mRNA, prostate collagen triple helix (PCOTH), transcript variant 1, mRNA, family with sequence similarity 124A (FAM124A), transcript variant 1, mRNA, interferon, alpha-inducible protein 27 (IFI27), transcript variant 2, mRNA, hypothetical protein MGC16121 (MGC16121), non-coding RNA, egf-like module containing, mucin-IMMORTA-GMPRC-PCT like, hormone receptor-like 2 (EMR2), transcript variant 4, mRNA, RAS guanyl releasing protein 2 (calcium and DAG-regulated) (RASGRP2), transcript variant 3, mRNA, KH domain containing, RNA binding, signal transduction associated 3 (KHDRBS3), mRNA, chromosome 9 open reading frame 47 (C9orf47), transcript variant 1, mRNA, family with sequence similarity 162, member B (FAM162B), mRNA, sodium channel, voltage-gated, type IX, alpha subunit (SCN9A), mRNA, glutaredoxin (thioltransferase) (GLRX), transcript variant 2, mRNA, four jointed box 1 (Drosophila) (FJX1), mRNA, chromosome 10 open reading frame 58 (C10orf58), transcript variant 1, mRNA, archaelysin family metallopeptidase 1 (AMZ1), mRNA, laminin, alpha 5 (LAMA5), mRNA, myosin XVI (MYO16), transcript variant 2, mRNA, SH3-domain GRB2-like (endophilin) interacting protein 1 (SGIP1), mRNA, nuclear receptor subfamily 2, group F, member 1 (NR2F1), mRNA, GULP, engulfment adaptor PTB domain containing 1 (GULP1), mRNA, actin binding LIM protein family, member 3 (ABLIM3), mRNA, transmembrane protein 154 (TMEM154), mRNA, germ cell associated 1 (GSG1), transcript variant 1, mRNA, suppressor of cytokine signaling 2 (SOCS2), mRNA, GULP, engulfment adaptor PTB domain containing 1 (GULP1), mRNA, integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), mRNA, DENN / MADD domain containing 2A (DENND2A), mRNA, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), transcript variant 2, mRNA, C-type lectin domain family 2, member B (CLEC2B), mRNA, hairy and enhancer of split 5 (Drosophila) (HES5), mRNA, chromosome 7 open reading frame 58 (C7orf58), transcript variant 2, mRNA, Keratin 18, cathepsin C (CTSC), transcript variant 1, mRNA, G protein-coupled receptor 183 (GPR183), mRNA, Mitogen-activated protein kinase kinase kinase 8 (MAP3K8), caspase recruitment domain family, member 9 (CARD9), transcript variant 2, mRNA, nidogen 1 (NID1), mRNA, adenosine monophosphate deaminase 3 (AMPD3), transcript variant 3, mRNA, opioid growth factor receptor-like 1 (OGFRL1), mRNA, interferon induced transmembrane protein 1 (9-27) (IFITM1), mRNA, interleukin 2 receptor, beta (IL2RB), mRNA, tripartite motif containing 14 (TRIM14), transcript variant 1, mRNA, acyl-CoA synthetase short-chain family member 1 (ACSS1), nuclear gene encoding mitochondrial protein, mRNA, RNA binding motif protein 24 (RBM24), transcript variant 3, mRNA, stathmin-like 2 (STMN2), transcript variant 1, mRNA, GULP, engulfment adaptor PTB domain containing 1 (GULP1), mRNA, secreted and transmembrane 1 (SECTM1), mRNA, betaine--homocysteine S-methyltransferase 2 (BHMT2), transcript variant 1, mRNA, lymphocyte-activation gene 3 (LAG3), mRNA,IMMORTA-GMPRC-PCT transmembrane protein 51 (TMEM51), transcript variant 1, mRNA, guanine nucleotide binding protein (G protein), gamma 11 (GNG11), mRNA, CD163 molecule-like 1 (CD163L1), mRNA, major histocompatibility complex, class I, F (HLA-F), transcript variant 2, mRNA, endoplasmic reticulum metallopeptidase 1 (ERMP1), mRNA, cytochrome b5 reductase 2 (CYB5R2), mRNA, mitogen-activated protein kinase kinase kinase 5 (MAP3K5), mRNA, interleukin 20 (IL20), mRNA, T-box 3 (TBX3), transcript variant 1, mRNA, adrenergic, alpha-1D-, receptor (ADRA1D), mRNA, leucine rich repeat containing 8 family, member C (LRRC8C), mRNA, folate receptor 3 (gamma) (FOLR3), mRNA, tumor necrosis factor receptor superfamily, member 21 (TNFRSF21), mRNA, actin binding LIM protein family, member 3 (ABLIM3), mRNA, CD44 molecule (Indian blood group) (CD44), transcript variant 7, mRNA, phosphatidylinositol-3,4,5- trisphosphate-dependent Rac exchange factor 1 (PREX1), mRNA, tripartite motif containing 14 (TRIM14), transcript variant 1, mRNA, aldehyde dehydrogenase 1 family, member A1 (ALDH1A1), mRNA, adaptor-related protein complex 1, mu 2 subunit (AP1M2), mRNA, integrin, alpha 1 (ITGA1), mRNA, mitogen-activated protein kinase kinase kinase 5 (MAP3K5), mRNA, serpin peptidase inhibitor, clade B (ovalbumin), member 2 (SERPINB2), transcript variant 2, mRNA, scavenger receptor class B, member 1 (SCARB1), transcript variant 1, mRNA, homeobox D9 (HOXD9), mRNA, G protein- coupled receptor, family C, group 5, member B (GPRC5B), mRNA, Rho guanine nucleotide exchange factor (GEF) 16 (ARHGEF16), mRNA, adrenergic, alpha-2A-, receptor (ADRA2A), mRNA, intercellular adhesion molecule 4 (Landsteiner-Wiener blood group) (ICAM4), transcript variant 2, mRNA, guanine nucleotide binding protein (G protein), gamma 4 (GNG4), transcript variant 1, mRNA, BCL2-like 10 (apoptosis facilitator) (BCL2L10), mRNA, monoglyceride lipase (MGLL), transcript variant 1, mRNA, Furry homolog (Drosophila) phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), mRNA, transmembrane and tetratricopeptide repeat containing 1 (TMTC1), transcript variant 1, mRNA, collagen, type IV, alpha 5 (COL4A5), transcript variant 1, mRNA, disrupted in renal carcinoma 3 (DIRC3), non-coding RNA, Rho GDP dissociation inhibitor (GDI) beta (ARHGDIB), mRNA, oxytocin receptor (OXTR), mRNA, tumor necrosis factor receptor superfamily, member 6b, decoy (TNFRSF6B), mRNA, cytochrome b5 reductase 2 (CYB5R2), mRNA, peptidase M20 domain containing 2 (PM20D2), mRNA, shroom family member 3 (SHROOM3), mRNA, family with sequence similarity 46, member C (FAM46C), mRNA, nuclear factor of kappa lightIMMORTA-GMPRC-PCT polypeptide gene enhancer in B-cells inhibitor, epsilon (NFKBIE), mRNA, tumor necrosis factor receptor superfamily, member 6b, decoy (TNFRSF6B), mRNA.

[0160] Without departing from the spirit of the invention, MSC, or pluripotent derived MSC may be optimized to possess heightened immune modulatory properties. In one embodiment this may be performed by exposure of MSC to hypoxic conditions, specifically hypoxic conditions can comprise an oxygen level of lower than 10%. In some embodiments, hypoxic conditions comprise up to about 7% oxygen. For example, hypoxic conditions can comprise up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, or up to about 1% oxygen. As another example, hypoxic conditions can comprise up to 7%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, or up to 1% oxygen. In some embodiments, hypoxic conditions comprise about 1% oxygen up to about 7% oxygen. For example, hypoxic conditions can comprise about 1% oxygen up to about 7% oxygen; about 2% oxygen up to about 7% oxygen; about 3% oxygen up to about 7% oxygen; about 4% oxygen up to about 7% oxygen; about 5% oxygen up to about 7% oxygen; or about 6% oxygen up to about 7% oxygen. As another example, hypoxic conditions can comprise 1% oxygen up to 7% oxygen; 2% oxygen up to 7% oxygen; 3% oxygen up to 7% oxygen; 4% oxygen up to 7% oxygen; 5% oxygen up to 7% oxygen; or 6% oxygen up to 7% oxygen. As another example, hypoxic conditions can comprise about 1% oxygen up to about 7% oxygen; about 1% oxygen up to about 6% oxygen; about 1% oxygen up to about 5% oxygen; about 1% oxygen up to about 4% oxygen; about 1% oxygen up to about 3% oxygen; or about 1% oxygen up to about 2% oxygen. As another example, hypoxic conditions can comprise 1% oxygen up to 7% oxygen; 1% oxygen up to 6% oxygen; 1% oxygen up to 5% oxygen; 1% oxygen up to 4% oxygen; 1% oxygen up to 3% oxygen; or 1% oxygen up to 2% oxygen. As another example, hypoxic conditions can comprise about 1% oxygen up to about 7% oxygen; about 2% oxygen up to about 6% oxygen; or about 3% oxygen up to about 5% oxygen. As another example, hypoxic conditions can comprise 1% oxygen up to 7% oxygen; 2% oxygen up to 6% oxygen; or 3% oxygen up to 5% oxygen. In some embodiments, hypoxic conditions can comprise no more than about 2% oxygen. For example, hypoxic conditions can comprise no more than 2% oxygen.

[0161] Enhancement of the wound healing activity of transfected MSC may be performed by altering the oxidative stress levels of the patient before, and / or during,IMMORTA-GMPRC-PCT and / or after administration of MSC. In one embodiment the patient is treated using MSC in combination with enhancing the antioxidant status of the patient. Enhancement of antioxidant status may be performed through administration of an antioxidant, or combination of antioxidants, said antioxidant may be selected from a group comprising of: ascorbic acid and derivatives thereof, alpha tocopherol and derivatives thereof, rutin, quercetin, allopurinol, hesperedin, lycopene, resveratrol, tetrahydrocurcumin, rosmarinic acid, Ellagic acid, chlorogenic acid, oleuropein, alpha-lipoic acid, glutathione, intravenous ascorbic acid, polyphenols, pycnogenol, retinoic acid, ACE Inhibitory Dipeptide Met-Tyr, recombinant superoxide dismutase, xenogenic superoxide dismutase, and superoxide dismutase.

[0162] In one embodiment, the nucleic acid introduced into the cell comprises an RNA. In another embodiment, at least one component of the CRISPR system comprises RNA. In yet another embodiment, the guide nucleic acid sequence is an RNA. In another embodiment, the RNA comprises in vitro transcribed RNA or synthetic RNA. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.

[0163] PCR can be used to generate a template for in vitro transcription of RNA which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5′ and 3′ UTRs. The primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5′ and 3 UTRs. Primers useful for PCR are generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region ofIMMORTA-GMPRC-PCT nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3′ to the DNA sequence to be amplified relative to the coding strand.

[0164] Chemical structures that have the ability to promote stability and / or translation efficiency of the RNA may also be used. The RNA preferably has 5′ and 3′ UTRs. In one embodiment, the 5′ UTR is between zero and 3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5′ and 3′ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0165] The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′ UTRs for the template. Alternatively, UTR sequences that are not endogenous for the template can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the template can be useful for modifying the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3′ UTR sequences can decrease the stability of RNA. Therefore, 3′ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0166] In one embodiment, the 5′ UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5′ UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5′ UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments the 5′ UTR can be derived from an RNA virusIMMORTA-GMPRC-PCT whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3′ or 5′ UTR to impede exonuclease degradation of the RNA.

[0167] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5′ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0168] In one embodiment, the mRNA has both a cap on the 5′ end and a 3′ poly(A) tail which determines ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3′ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.

[0169] On a linear DNA template, phase T7 RNA polymerase can extend the 3′ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0170] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However, polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA / T 3′ stretch without cloning highly desirable.

[0171] The polyA / T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size canIMMORTA-GMPRC-PCT be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.

[0172] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E- PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3′ end can increase mRNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0173] 5′ caps also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5′ cap. The 5′ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0174] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.

[0175] The RNAs described herein may be introduced into the cell by a variety of methods known in the art. In some embodiments, the RNA is electroporated into the cells. In one embodiment, the CRISPR system comprises an RNA that is electroporated into the cells. In yet another embodiment, the CRISPR system comprises at least one guide nucleic acid sequence that is an RNA and electroporated into the cells.IMMORTA-GMPRC-PCT

[0176] The disclosed methods can be applied to the modulation of cell activity in order to provide therapy to the subject in the fields of cancer, acute and chronic infections, and autoimmune diseases. The disclosed methods can involve targeting stem cells, and also can include methods for assessing the ability of the genetically modified cell to kill a target cancer cell.

[0177] The methods also provide the ability to control the level of expression over a wide range by changing, for example, the promoter or the amount of input RNA, making it possible to individually regulate the expression level.

[0178] One advantage of RNA transfection methods of the invention is that RNA transfection is essentially transient and a vector-free. An RNA transgene can be delivered to a cell and expressed therein, as a minimal expressing cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cloning of cells is not necessary because of the efficiency of transfection of the RNA and its ability to uniformly modify the entire lymphocyte population.

[0179] Genetic modification of the cells with in vitro-transcribed RNA (IVT-RNA) makes use of two different strategies both of which have been successively tested in various animal models. Cells are transfected with in vitro-transcribed RNA by means of lipofection or electroporation. It is desirable to stabilize IVT-RNA using various modifications in order to achieve prolonged expression of transferred IVT-RNA.

[0180] Another embodiment disclosed herein is to exponentially expand the cells of the invention such as personalized regenerative cells and derivatives thereof using hollow fiber bioreactors. Human cells have been grown in hollow fiber bioreactors for many decades and offer advantages as a closed system with better safety from micro-organism contamination and from reduced human error. The hollow fiber media would continue to be CD / XF media with E10 media supplemented with other anti-cancer small molecules described below to expand to about 100 million to 1000 million or more cells.

[0181] As another embodiment of the disclosure, E10 media is further enhanced for stem cell maintenance and self-renewal during rapid expansion by maintaining some EPR drug treatments using at least one of the EPR drugs at the following doses: 1-3 μM Forskolin and 1-3 μM GSK3 inhibitor CHIR99021, 1-5 μM ROCK inhibitor Y-27632,IMMORTA-GMPRC-PCT and 1-5 μM Gö6983 targeting Protein Kinase C (PKC). The PKC drug Gö6983 is also an important biochemical pathway in controlling cancer

[0024] . The enhanced E10 with the EPR inhibitors is denoted as media E12 in the current disclosure. Thus, stem cell expansion will typically be done in E12 media with at least one EPR drugs.

[0182] Another embodiment of the disclosure is to reduce cancer risks during ex vivo adipose stem cell (ASC) expansion by adding a group of 3 to 6 anti-tumor botanicals (Anti-Tumor Additives or ATA) to the E12 media. The components of ATA include at least 3 of the following 6 chemically defined additives: Astragaloside IV, Apigenin, Berberine, Fisetin, Genistein, and Lithium Orotate. All the selected ATA botanicals have shown anticancer activity in multiple published studies. The doses in tissue culture for each of the ATA components comprise: 20 to 100 nM Astragaloside IV; 2 to 10 μM Apigenin; 20 to 100 μM Berberine; 10 to 40 μM Fisetin; 20 to 100 nM Genistein; and 2 to 10 μM Lithium Orotate. These six botanical products kill or slow the growth of cancer cells while having little or no effects on the growth of non-cancerous cells. Cell expansion will typically be done in E12 media with supplemented with the 3-6 antitumor additives (ATA).

[0183] Another embodiment of the current disclosure is to use the cell expansion systems above to exponentially expand MSCs from cord blood or any other non- cancerous source. We have successfully expanded adipose-derived MSCs from a 60 year old man without reprogramming his stem cells, so the general expansion systems work with aged adult stem cells that have not been rejuvenated. Our cell expansion systems could thus be used to expand cord blood MSCs, which do not contain enough stem cells for adults.

[0184] As another embodiment of the current disclosure, the final expanded populations of cells of the invention comprising personalized regenerative cells or derivatives thereof are tested for methylated DNA age and cancer-free tissue function before injecting back into the mammal. The expanded population of cells is tested to ensure that the mean epigenetic age is like fetal MSCs and that the cell gene expression profile remains similar to the pattern found in young fetal MSCs from fetal blood, bone marrow, or adipose tissue. T cells are also tested for the contamination with cancer stem cells and teratomas by verifying that the cells do not form cancer when injected into JAX® Nude Mice. Cell function is also tested by their ability to form human bone matrix,IMMORTA-GMPRC-PCT connective fiber, muscle fiber, kidney tissue, heart muscle, and neurons when injected into the appropriate tissues in JAX® Nude Mice. The cells that pass all these quality tests plus tests for bacterial or viral infections can then safely be used clinically in the mammal. In certain embodiments, approximately 0.05 to 1.0 billion cells expanded and tested cells can be injected into an organ of the mammal to improve organ function or injected systemically into the circulatory system or bone marrow of the mammal to reduce disease conditions or mortality.

[0185] As another embodiment of the current disclosure, a sample of expanded and tested cells generated using the methods of the invention are frozen in liquid nitrogen in numerous tubes with a million or more cells per tube as renewable stocks of cells as the mammal ages. Since the cells are a valuable autologous stem cell resource that is easily expanded for later use, the cells will be banked in frozen storage for use in the decades after their generation. In this respect, frozen cell stocks would be like having your own frozen umbilical cord stem cells that have proven to be very helpful to the lucky few whose parents thought to save what is typically thrown away on birthing. However, umbilical cord stem cells often only contain enough stem cells to treat up to a 10-year-old child. Using the general conditions outlined in this disclosure, we have expanded stocks of personalized regenerative cells over a million-fold and 42 passages with no significant increases in epigenetic age as determined by DNA methylation. A frozen vial of the adult stem cells can be thawed and expanded ex vivo to some 1000 million cells under the same ex vivo regiment at 3% oxygen and 5% carbon dioxide in low glucose DMEM / F12 with the same defined and xeno-free media containing the anti-tumor additives. Cell frozen stocks prepared by the specified system in the current disclosure can be a major game changer for employing safe functional fetal-like stem cells to treat many differing conditions even in aged individuals.

[0186] As another embodiment of the current disclosure, longevity genes can be edited into cells such as reprogrammed autologous cells using CRISPR or other older gene targeted techniques. One such gene is the oncogene NCORE (a corepressor of histone acetylase), which can be mutated to prevent the inhibition of apoptosis or cell suicide, which helps prevent the generation of metastatic cancer cells and helps kill off dysfunctional senescent cells. Another is the Growth Hormone Receptor (GHR), which is often mutated in individuals that live over 100 years. Another favored mutated gene is theIMMORTA-GMPRC-PCT Insulin-Like Growth Factor receptor (IGF-R), which is often found in long lived women. To take advantage of these beneficial gene modification to a mammal, a mammal's ASCs are rejuvenated into cells as described above. Various genes (e.g., NORE, GHR, or IGF- R) in the cells are then gene edited using CHRISR or other older gene targeting techniques under the same ex vivo regiment of low glucose DMEM / F12 with E10 media. The edited cells are then cultured for 7 to 21 days in DMEM / F12 with E12 media and individual cell clones isolated and assayed to check for correct gene editing. The RAN cell clones with the correct gene edits are expanded exponentially in the hollow fiber bioreactors as described above.

[0187] In another embodiment of the disclosure, the systems herein can also be useful in gene editing of human patients with rare genetic diseases. Using viral vectors to edit the human patients' cells in the body is challenging. Using the technology described herein, one can do the gene editing in vitro using CRISPR after reprogramming the ASCs to therapeutic cells of the invention. The edited cells that removes the genetic mutation are then cultured in DMEM / F12 and individual cell clones isolated and assayed to check for correct mutational editing. The cell clones with the correct gene edits are then expanded in the hollow fiber bioreactors as described above. The mutant-edited cells can then be injected back into the patient to treat their specific genetic disease. The gene edited cell system circumvents the need to do gene editing of the patient's cells in vivo, which is inherently risky. Using the techniques in the current disclosure also benefits systems that use gene editing in vitro, as the patient's edited cells can be cloned easily and expanded greatly before injection into the patient. In this case, multiple injection treatments with rejuvenated fully functional cells should greatly increase the chances of success with in vitro edited cells. Some IVT vectors are known in the literature which are utilized in a standardized manner as template for in vitro transcription and which have been genetically modified in such a way that stabilized RNA transcripts are produced. Currently protocols used in the art are based on a plasmid vector with the following structure: a 5′ RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3′ and / or 5′ by untranslated regions (UTR), and a 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette by type II restriction enzymes (recognition sequence corresponds to cleavage site). The polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of thisIMMORTA-GMPRC-PCT procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and extend or mask the poly(A) sequence at the 3′ end. It is not clear, whether this non- physiological overhang affects the amount of protein produced intracellularly from such a construct.

[0188] In another aspect, the RNA construct is delivered into the cells by electroporation. See, e.g., the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1. The various parameters including electric field strength required for electroporation of any known cell type are generally known in the relevant research literature as well as numerous patents and applications in the field. See e.g., U.S. Pat. No.6,678,556, U.S. Pat. No.7,171,264, and U.S. Pat. No.7,173,116. Apparatus for therapeutic application of electroporation is available commercially, e.g., the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Pat. No.6,567,694, U.S. Pat. No.6,516,223, U.S. Pat. No.5,993,434, U.S. Pat. No.6,181,964, U.S. Pat. No.6,241,701, and U.S. Pat. No.6,233,482; electroporation may also be used for transfection of cells in vitro as described, e.g., in US20070128708A1. Electroporation may also be utilized to deliver nucleic acids into cells in vitro. Accordingly, electroporation-mediated administration into cells of nucleic acids including expression constructs utilizing any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new means for delivering an RNA of interest to a target cell.

[0189] Pharmaceutical compositions of the present invention may comprise the modified cell as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.

[0190] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity andIMMORTA-GMPRC-PCT frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0191] When “an immunologically effective amount”, “an anti-immune response effective amount”, “an immune response-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, immune response, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the modified cells described herein may be administered at a dosage of 104 to 109 cells / kg body weight, preferably 105 to 106 cells / kg body weight, including all integer values within those ranges. Cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319, 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0192] In certain embodiments of the present invention, the cells are expanded and modified using the methods described herein, or other methods known in the art where the cells are expanded to therapeutic levels, and administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efalizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the modified cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al., Cell 66:807-IMMORTA-GMPRC-PCT 815, 1991; Henderson et al., Immun.73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

[0193] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art- accepted practices. The dose for CAMPATH, for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day although in some instances, larger doses of up to 40 mg per day may be used (described in U.S. Pat. No.6,120,766).

[0194] It should be understood that the method and compositions that would be useful in the present invention are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0195] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010);IMMORTA-GMPRC-PCT “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention.

Claims

IMMORTA-GMPRC-PCT WHAT IS CLAIMED IS:

1. A method for treating a subject with a reprogrammed autologous cell, the method comprising: a) identifying a human subject with a disease; b) providing an in vitro reprogrammed autologous cell, wherein the cell comprises a vector, wherein the vector comprises a polynucleotide encoding a gene switch, and wherein the gene switch comprises the following: i) at least one transcription factor sequence that encodes a ligand-dependent transcription factor comprising an ecdysone receptor ligand binding domain, operably linked to a promoter; and ii) a polynucleotide encoding a therapeutic polypeptide sequence linked to a promoter that is activated by said ligand- dependent transcription factor, wherein the therapeutic polypeptide sequence is at least 85% identical to the wild type human polypeptide sequence; c) administering the in vitro reprogrammed autologous cell to the subject; and d) administering the ligand to the subject.

2. The method of Claim 1, wherein a reprogrammed autologous cell is derived from an induced pluripotent stem cell.

3. The method of Claim 1, wherein a reprogrammed autologous cell expresses a marker selected from the group consisting of: CD41a, CD44, CD90, and CD105, NeuN, CD9, CD62, CD59, Actin, GFAP, NSE, Nestin, CD73, SSEA-4, hTERT, Oct-4, and tubulin.

4. The method of Claim 1, wherein a reprogrammed autologous cell is capable of differentiating into a cell lineage selected from the group consisting of: adipogenic, endothelial, hepatic, osteogenic, neural, pancreatic, and myocytic.

5. The method of Claim 1, wherein the vector is selected from the group consisting of: lentiviral vector, adenoviral vector, and adeno-associated viral vector.

6. The method of Claim 1, wherein the polynucleotide encoding a gene switch comprises a first transcription factor sequence and a second transcription factor sequence, wherein the first and second transcription factor sequences may be under control of the same or different promoters, and wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand- dependent transcription factor.IMMORTA-GMPRC-PCT 7. The method of Claim 1, wherein the ligand is selected from the group consisting of RG-115819, RG-115932, and RG-115830.

8. The method of Claim 1, wherein the ligand is selected from the group consisting of: an amidoketone, an oxadiazoline, and a diacylhydrazine.

9. The method of Claim 1, wherein the ligand is administered to the subject less than 1 hour, less than 24 hours, or less than 48 hours following administration of the reprogrammed autologous cell.

10. The method of Claim 1, wherein the ligand is administered to the subject daily for a period of at least 2 days, at least 7 days, at least 10 days, at least 14 days, or at least 30 days beginning on the day that the reprogrammed autologous cells are administered to the subject.

11. The method of Claim 1, wherein the polynucleotide encoding a gene switch comprises a sequence encoding a domain selected from the group consisting of: a VP-16 transactivation domain, a GAL-4 DNA binding domain, and a RXR ligand binding domain.

12. The method of Claim 1, wherein administering the in vitro reprogrammed autologous cell and the ligand induces an immune response against a tumor in the subject.

13. The method of Claim 1, wherein the polynucleotide encoding a gene switch comprises the following: i) a first transcription factor sequence encoding a VP-16 transactivation domain and a chimeric RXR ligand binding domain; ii) an EMCV IRES; and iii) a second transcription factor sequence encoding a GAL4 DNA- binding domain and a Choristoneura fumiferana ligand binding domain comprising a substitution mutation, and wherein the ligand is a diacylhydrazine.

14. The method of Claim 1, wherein in vivo persistence of the autologous cell is augmented by modifying apoptosis of the cell by a method comprising: i) transfecting the cell with a gene construct encoding an anti-apoptotic gene; or ii) transfecting the cell with a gene construct capable of inducing RNA interference directed against a molecule associated with induction of apoptosis.

15. The method of Claim 14, wherein the anti-apoptotic gene is selected from the group consisting of obestatin, XIAP, sphingosine-1-phosphate, BCL-2, BCL-XL, GATA-4, IGF-1, EGF, heme-oxygenase-1, NF-kB, akt, pi3-k, and epha-2.IMMORTA-GMPRC-PCT 16. The method of Claim 14, wherein the molecule associated with induction of apoptosis is selected from the group consisting of: Fas, FasL, CASP1 (ICE), CASP10 (MCH4), CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR (CASPER), CRADD, PYCARD (TMS1 / ASC), ABL1, AKT1, BAD, BAK1, BAX, BCL2L11, BCLAF1, BID, BIK, BNIP3, BNIP3L, CASP1 (ICE), CASP10 (MCH4), CASP14, CASP2, CASP4, CASP6, CASP8, CD70 (TNFSF7), CIDEB, CRADD, FADD, FASLG (TNFSF6), HRK, LTA (TNFB), NOD1 (CARD4), PYCARD (TMS1 / ASC), RIPK2, TNF, TNFRSF10A, TNFRSF10B (DR5), TNFRSF25 (DR3), TNFRSF9, TNFSF10 (TRAIL), TNFSF8, TP53, TP53BP2, TRADD, TRAF2, TRAF3, and TRAF4.

17. The method of Claim 1, wherein the in vitro reprogrammed autologous cell is induced to differentiate into a neural lineage cell, and wherein differentiation into the neural lineage cell is conferred by transfecting the cell with a polynucleotide encoding a therapeutic polypeptide selected from the group consisting of: ADCYAP1R1, ARTN, BDNF, CD40 (TNFRSF5), CNTF, CNTFR, CRHBP, CRHR1, CRHR2, FRS2, FRS3, FUS, GDNF, GFRA1, GFRA2, GFRA3, GMFB, GMFG, MAGED1, MT3, NF1, NGF, NGFR, NGFRAP1, NR1I2, NRG1, NRG2, NTF3, NTF4, NTRK1, NTRK2, PSPN, PTGER2, TFG, TRO, and VGF.

18. The method of Claim 1, wherein the in vitro reprogrammed autologous cell is endowed with the ability to migrate or home to a tissue or a tumor site, and wherein migration and homing ability is conferred by transfecting the cell with a polynucleotide sequence encoding a therapeutic polypeptide selected from the group consisting of: SDF-1, VEGF, FGF-1, FGF-2, PDGF-BB, angiopoietin, and TSPL-1.

19. The method of Claim 1, wherein the in vitro reprogrammed autologous cell is endowed with the ability to stimulate angiogenesis, and wherein angiogenic ability is conferred by transfecting the cell with a polynucleotide encoding a therapeutic polypeptide selected from the group consisting of: activin A, adrenomedullin, aFGF, ALK1, ALK5, ANF, angiogenin, angiopoietin-1, angiopoietin-2, angiopoietin-3, angiopoietin-4, bFGF, B61, bFGF inducing activity, cadherins, CAM-RF, cGMP analogs, ChDI, CLAF, claudins, collagen, collagen receptors .alpha..sub.1.beta..sub.1 and .alpha..sub.2.beta..sub.1, connexins, Cox-2, ECDGF (endothelial cell-derived growth factor), ECG, ECI, EDM, EGF, EMAP, endoglin, endothelins, endostatin, endothelial cell growthIMMORTA-GMPRC-PCT inhibitor, endothelial cell-viability maintaining factor, endothelial differentiation sphingolipid G-protein coupled receptor-1 (EDG1), ephrins, Epo, HGF, TGF-beta, PD-ECGF, PDGF, IGF, IL8, growth hormone, fibrin fragment E, FGF-5, fibronectin and fibronectin receptor .alpha.5.beta.1, Factor X, HB-EGF, HBNF, HGF, HUAF, heart derived inhibitor of vascular cell proliferation, IL1, IGF-2 IFN-gamma, integrin receptors, K-FGF, LIF, leiomyoma-derived growth factor, MCP-1, macrophage-derived growth factor, monocyte-derived growth factor, MD-ECI, MECIF, MMP 2, MMP3, MMP9, urokinase plasminogen activator, neuropilin (NRP1, NRP2), neurothelin, nitric oxide donors, nitric oxide synthases (NOSs), notch, occludins, zona occludins, oncostatin M, PDGF, PDGF-B, PDGF receptors, PDGFR-.beta., PD-ECGF, PAI-2, PD-ECGF, PF4, P1GF, PKR1, PKR2, PPAR-gamma, PPAR-gamma ligands, phosphodiesterase, prolactin, prostacyclin, protein S, smooth muscle cell-derived growth factor, smooth muscle cell-derived migration factor, sphingosine-1-phosphate-1 (SIP1), Syk, SLP76, tachykinins, TGF-beta, Tie 1, Tie2, TGF-.beta., and TGF-.beta. receptors, TIMPs, TNF-alpha transferrin, thrombospondin, urokinase, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF.sub.164, VEGI, and EG-VEGF.

20. The method of Claim 1, wherein the in vitro reprogrammed autologous cell is endowed with the ability to stimulate an immune response, and wherein immune stimulatory ability is conferred by transfecting the cell with a polynucleotide encoding a therapeutic polypeptide selected from the group consisting of: ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, IFNA2, IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, CD40LG (TNFSF5), IFNA2, , IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, IL22, IL5, IL-6, IL8, IL9, IL-18, IL-33, LTA, LTB, MIF, SCYE1, SPP1, TNF, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), C5, CCL1 (I- 309), CCL11 (eotaxin), HMGB1, IL-2. IL-12, IL-17, IL33. CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), complement components C3, and C5, 2,3 alpha gal, CCL21 (MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2 / eotaxin-2), CCL25 (TECK) , CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (I-TAC / IP-9),IMMORTA-GMPRC-PCT CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78 / LIX), CXCL6 (GCP-2), CXCL9, IL13, and IL8.