WO2011035018A2

WO2011035018A2 - Suicide ready cells

Info

Publication number: WO2011035018A2
Application number: PCT/US2010/049112
Authority: WO
Inventors: John D. Mendlein; Paul Grayson
Original assignee: Fate Therapeutics, Inc.
Priority date: 2009-09-18
Filing date: 2010-09-16
Publication date: 2011-03-24
Also published as: WO2011035018A3

Abstract

The present invention includes methods of providing treatment to individuals in need of cell, tissue, and/or organ repair, regeneration, restoration, and/or replacement by administering cell-based compositions. The cell-based compositions of the present invention have features that increase both the safety and therapeutic efficacy of regenerative therapy. The present invention also includes implants that can be used to provide regenerative therapy.

Description

SUICIDE READY CELLS

BACKGROUND Technical Field

The present invention generally relates to providing a cell-based composition to individuals in need of cell, tissue, and/or organ repair, regeneration, restoration, and/or replacement. More particularly, the present invention generally relates to providing cell-based compositions comprising features that increase both the safety and therapeutic efficacy of regenerative therapy. Description of the Related Art

There is tremendous potential, albeit unrealized, for cell based therapies and gene therapy to change the face of modern medicine.

Regenerative medicine has the potential to repair, replace, or regenerate any tissue. However, in order for the field of regenerative therapy to more fully realize its potential, substantial obstacles routinely encountered or envisaged with stem cell-based therapies must be overcome.

In addition to the moral and ethical issues involved with using embryos, safety is an important factor to be considered when discussing the benefits of different types of stem cells therapies.

The use of embryonic stem cell therapies faces a number of practical issues, including, but not limited to microbial contamination or other foreign contaminants, genetic instability, and risk of oncogenesis. In addition, certain embryonic stem cell lines approved for research are no longer "pure" human stem cell lines, in part, because the hESCs are exposed to and/or cultured on mouse fibroblast cells or "feeder cells" in order to grow, maintain, and/or expand hESCs. Studies have also indicated that the developmental potency of these "older" approved hESC lines is also increasingly compromised by time in culture, number of passages and freeze/thaw cycles. These older hESC lines also accumulate genetic abnormalities as they replicate. Rapid replication of hESC with genetic instability may create a risk of tumor

development in both animal and human transplants.

Graft versus host disease is also a substantial risk in fetal stem cell and adult stem cell therapies. Fetal/adult stem cells may be contaminated with blood and other tissue cells which have developed immune defenses to "foreign" cells. These fetal cells may cause immune reactions and severe health problems in a patient receiving them. In some instances,

pharmaceuticals that reduce immune reactivity are used in concert with the cell- based therapies. However, oftentimes such pharmaceutical intervention promotes unwanted side effect of being toxic to the transplanted cells. For example, in neural stem cell transplants, sometimes cortisone is administered to decrease the immune response to the transplant. However, cortisone can elevate glutamate levels to toxic levels for the neural stem cells and thus, can compromise the effectiveness of treatments for brain injuries and disorders.

An important element in assessing stem cell safety is to answer the question: do stem cells act as they are intended once transplanted? The unpredictable reality may be that once implanted, stem cells begin to

uncontrollably divide and differentiate into cancer cells, leading to tumor growth. To control for these possibilities, scientists test the therapies by inducing conditions in laboratory animals. However, laboratory animal models are still only baseline predictors of how stem cells may behave once transplanted in humans. Because unregulated growth of transplanted hESCs may result in tumor formation, alternative strategies are needed before stem cells can reach mainstream status for use to treat the full range of diseases they show potential to treat.

Thus, there is a significant and unmet need for identifying approaches by which cells, including stem cells and progenitor cells, can be more efficiently used in cell-based compositions and regenerative therapy. The present invention addresses these needs and offers other related advantages. BRIEF SUMMARY

In various embodiments, the present invention provides, in part, a method of cell-based therapy comprising: administering to the individual, a plurality of genetically modified cells and one or more cytotoxic agents, wherein the cells are resistant to the cytotoxic agent; allowing the cells to remain at the site under conditions and for a time sufficient to provide an amount of therapy to the individual; and inducing apoptosis in the cells, thereby providing cell-based therapy.

In a particular embodiment, the cells are directed to a site in vivo. In another particular embodiment, the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell.

In yet another particular embodiment, the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: a cell differentiated from an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell.

In certain embodiment, the plurality of genetically modified cells comprises at least one somatic cell.

In related embodiments, the plurality of genetically modified cells comprises a cell isolated from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, and kidney.

In other related embodiments, the plurality of genetically modified cells comprises a mammalian cell.

In still yet other related embodiment, the plurality of genetically modified cells comprises a human cell.

In another embodiment, each of the plurality of genetically modified cells comprises at least one resistance transgene that provides resistance to the one or more cytotoxic agents. In a particular embodiment, the at least one resistance transgene encodes a polypeptide selected from the group consisting of: ABCG2, BCRP, LRP, MRP1 , MRP2, MRP3, MRP4, MRP5, MRP6, MRP8, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S-transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine

deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2, thrombopoietin receptor F36Vmpl, IRDS (interferons), HREP450R, SIRT1 , and variants, and functional fragments thereof.

In a certain embodiment, each of the plurality of genetically modified cells comprises at least one cell suicide transgene that is capable of inducing the apoptosis of said cells.

In a certain related embodiment, at least one cell suicide transgene encodes a polypeptide selected from the group consisting of:

caspase-3, caspase-8, caspase-9, caspase-12, apoptosis inducing factor, BAD, and BIM.

In another certain related embodiment, the at least one cell suicide transgene encodes a polypeptide selected from the group consisting of: thymidine kinase, HSVTK39, carboxylesterase , carboxypeptidase G2, cytochrome P450, cytosine deaminase, nitroreductase 1 , FAS-FK506 binding protein (FKBP) fusion protein, FADD-FK506 fusion protein, caspase-3 death domain-FK506 fusion protein, caspase-6 death domain-FK506 fusion protein, caspase-7 death domain-FK506 fusion protein, caspase-8 death domain-FK506 fusion protein, caspase-9 death domain-FK506 fusion protein, caspase-10 death domain-FK506 fusion protein, CD20, novel EC domains, and novel EC domains fused to chemical dimerization domain and intracellular death domain.

In a further embodiment, apoptosis is induced by contacting the plurality of genetically modified cells with a small molecule.

In another embodiment, the small molecule is converted into a cytotoxic agent that induces apoptosis in the genetically modified cells.

In yet another embodiment, the small molecule allows the multimerization of polypeptides that induce apoptosis upon multimerization. In a particular embodiment, the at least one cell suicide transgene encodes an RNAi molecule directed against a gene encoding a polypeptide that is essential for cell survival.

In another particular embodiment, the gene that encodes a polypeptide that is essential for cell survival is selected from the group consisting of: survivin, IAP, and Bcl2.

In one embodiment, the site in vivo is selected from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, or kidney.

In another embodiment, the one or more cytotoxic agents are selected from the group consisting of: chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine,

trietylenephosphoramide, triethiylenethiophosphoramide and

trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone;

lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-1 1 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin;

callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1 -TM1 ); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfanide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,

novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;

nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammal I and calicheamicin omegaH (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin

chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,

cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,

cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCI liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine

(GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin,

methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;

pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;

procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotnethylannine; trichothecenes (especially T-2 toxin, verracurin A, rohdin A and anguidine); urethan; vindesine (ELDISINE®,

FILDESIN®); dacarbazine; mannonnustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;

methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; amninopterin; cyclosporine, sirolimus, rapamycin, ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine

(DMFO); retinoids such as retinoic acid; CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin

(ELOXATIN™) combined with 5-FU, leucovovin; anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene, 4- hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and

toremifene (FARESTON®); anti-progesterones; estrogen receptor down- regulators (ERDs); estrogen receptor antagonists such as fulvestrant

(FASLODEX®); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMAS IN®), formestanie, fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate

(DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); aptamers, described for example in U.S. Patent No. 6,344,321 , which is herein incorporated by reference in its entirety; anti HGF monoclonal antibodies {e.g., AV299 from Aveo, AMG102, from Amgen); truncated c-Met variants {e.g., CGEN241 from Compugen); protein kinase inhibitors that block c-Met induced pathways {e.g., ARQ197 from Arqule, XL880 from Exelexis, SGX523 from SGX Pharmaceuticals, MP470 from Supergen, PF2341066 from Pfizer); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor {e.g., LURTOTECAN®); rmRH {e.g.,

ABARELIX®); lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); COX-2 inhibitors such as celecoxib (CELEBREX®; 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1 H-pyrazol- 1 -yl) benzenesulfonamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In certain embodiments, at least one of the plurality of the genetically modified cells remains at the site for about 1 week to about 1 year; for about 1 month to about 1 year; for about 6 months to about 1 year; for about 1 year; or for more than 1 year.

In a particular embodiment, each of the genetically modified cells comprises at least one factor transgene that encodes a dedifferentiation factor, a differentiation factor, a growth factor or a cytokine.

In another embodiment, the factor transgene encodes at least the dedifferentiation factors Oct ¾, Sox 2, and Nanog. In a related embodiment, the factor transgene encodes the dedifferentiation factor Oct ¾.

In a certain embodiment, the factor transgene encodes a growth factor or cytokine selected from the group consisting of: brain derived neurotropihic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (BMP-2), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 -CCL28, CXCL1 -CXCL17, XCL1 , XCL2, CX3CL1 , vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF), FAS-ligand, fibroblast growth factor 1 (FGF-1 ), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 (FGF-10), Flt-3, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM-CSF), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN-b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1 ), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 1 1 (IL-1 1 ), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 19 (IL-19), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derived growth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF- CC), platelet derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell derived factor 1 (SDF-1 ), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), Wnt1 , Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt1 1 , Wnt14, Wnt15, or Wnt16, Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

In other embodiments, expression of one or more transgenes is inducible.

In yet other embodiment, expression of one or more transgenes is constitutive. In one embodiment, a plurality of non-genetically modified cells is also administered to the individual. In a particular embodiment, the plurality of non-genetically modified cells is selected from the group consisting of: isolated adult stem cells, isolated embryonic stem cells, induced pluripotent stem cells, and cells differentiated therefrom.

In another particular embodiment, the non-genetically modified cells are administered prior to the genetically modified cells. In a certain embodiment, the non-genetically modified cells are administered at

substantially the same time as the genetically modified cells. In certain particular embodiments, the genetically modified cells are administered prior to the non-genetically modified cells.

In various other embodiment, the present invention also provides, in part, a kit comprising: one or more cytotoxic agents that target at least one population of susceptible cells; a plurality of genetically modified cells comprising at least one resistance transgene that provides resistance to the one or more cytotoxic agents and at least one inducible cell suicide transgene; and a pharmaceutically acceptable cell culture medium.

In one embodiment, the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell.

In another embodiment, the plurality of genetically modified cells comprises a cell differentiated from an isolated adult stem cell, an isolated embryonic stem cell, or an induced pluripotent stem cell.

In yet another embodiment, the plurality of genetically modified cells comprises a somatic cell.

In particular embodiments, the plurality of genetically modified cells is isolated from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, hematopoietic cells, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, and kidney. In other particular embodiments, the plurality of genetically modified cells is mammalian. In yet other particular embodiment, the plurality of genetically modified cells is human.

In one embodiment, the kit comprises a plurality of non-genetically modified cells.

In a particular embodiment, the plurality of non-genetically modified cells is selected from the group consisting of: isolated adult stem cells, isolated embryonic stem cells, induced pluripotent stem cells, and cells differentiated therefrom.

In a certain embodiment, the at least one resistance transgene that provides resistance to one or more cytotoxic agents encodes a polypeptide selected from the group consisting of: ABCG2, BCRP, LRP, MRP1 , MRP2, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S- transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2, thrombopoietin receptor F36Vmpl, IRDS (interferons), HREP450R, SIRT1 , and variants, and functional fragments thereof.

In another certain embodiment, the at least one inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: caspase-3, caspase-8, caspase-9, caspase-12, apoptosis inducing factor, BAD, and BIM.

In a certain related embodiment, the at least one cell inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: thymidine kinase, HSVTK39, carboxylesterase , carboxypeptidase G2, cytochrome P450, cytosine deaminase, nitroreductase 1 , FAS-FK506 binding protein (FKBP) fusion protein, FADD-FK506 fusion protein, caspase-3 death domain-FK506 fusion protein, caspase-6 death domain-FK506 fusion protein, caspase-7 death domain-FK506 fusion protein, caspase-8 death domain-FK506 fusion protein, caspase-9 death domain-FK506 fusion protein, caspase-10 death domain-FK506 fusion protein, CD20, novel EC domains, and novel EC domains fused to chemical dimerization domain and intracellular death domain. In an additional embodiment, a kit comprises a small molecule that induces apoptosis by contacting the plurality of genetically modified cells. In another embodiment, the small molecule is converted into a cytotoxic agent that induces apoptosis in the genetically modified cells. In yet another embodiment, the small molecule allows the multimerization of polypeptides that induce apoptosis upon multimerization.

In a further embodiment, the at least one cell suicide transgene encodes an RNAi molecule directed against a gene encoding a polypeptide that is essential for cell survival.

In a particular embodiment, the gene that encodes a polypeptide that is essential for cell survival is selected from the group consisting of:

survivin, IAP, and Bcl2.

In one embodiment, the plurality of genetically modified cells is capable of being directed to a site in vivo, wherein the site is selected from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, or kidney.

trietylenephosphoramide, triethiylenethiophosphoramide and

callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1 -TM1 ); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfanide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;

nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammal I and calicheamicin omegaH (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin

cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCI liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopterin,

pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;

procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;

triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®,

FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and doxetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine;

(FASLODEX®); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMASIN®), formestanie, fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate

In a particular embodiment, each of the genetically modified cells comprises at least one factor transgene that encodes a dedifferentiation factor, a differentiation factor, a growth factor, a cytokine, or a hormone.

In another particular embodiment, at least one dedifferentiation factor polypeptide, differentiation factor polypeptide, growth factor polypeptide, cytokine, or hormone. In another embodiment, a kit comprises one or more

dedifferentiation agents, differentiation agents, or agents that increase cell growth and/or proliferation.

In particular embodiments, expression of one or more transgenes is inducible. In other particular embodiments, expression of one or more transgenes is constitutive.

In certain embodiments, a kit comprises a biocompatible matrix. In other certain embodiments, a kit comprises an implant.

In various other embodiments, the present invention provides, in part, an implant comprising: one or more cytotoxic agents; a composition comprising a plurality of genetically modified cells, said cells comprising one or more resistance transgenes that provide resistance to the one or more cytotoxic agents and one or more inducible cell suicide transgenes; a biocompatible matrix; and a pharmaceutically acceptable cell culture medium.

In one embodiment the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell. In a related embodiment, the plurality of genetically modified cells comprises a cell differentiated from an isolated adult stem cell, an isolated embryonic stem cell, or an induced pluripotent stem cell. In yet another related embodiment, the plurality of genetically modified cells comprises a somatic cell.

In particular embodiments, the plurality of genetically modified cells is isolated from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, hematopoietic cells, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, and kidney.

In other particular embodiments, the plurality of genetically modified cells is mammalian.

In yet other particular embodiments, the plurality of genetically modified cells is human.

In a certain embodiment, an implant comprises a plurality of non- genetically modified cells. In a certain related embodiment, the plurality of non- genetically modified cells is selected from the group consisting of: isolated adult stem cells, isolated embryonic stem cells, induced pluripotent stem cells, and cells differentiated therefrom.

In an additional embodiment, at least one resistance transgene that provides resistance to one or more cytotoxic agents encodes a polypeptide selected from the group consisting of: ABCG2, BCRP, LRP, MRP1 , MRP2, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S- transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2, thrombopoietin receptor F36Vmpl, IRDS (interferons), HREP450R, SIRT1 , and variants, and functional fragments thereof.

In one embodiment, the at least one inducible cell suicide transgene encodes a polypeptide selected from the group consisting of:

caspase-3, caspase-9, apoptosis inducing factor, BAD, and BIM.

In a particular embodiment, the at least one cell inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: thymidine kinase, HSVTK39, carboxylesterase , carboxypeptidase G2, cytochrome P450, cytosine deaminase, nitroreductase 1 , FAS-FK506 binding protein (FKBP) fusion protein, FADD-FK506 fusion protein, caspase-3 death domain-FK506 fusion protein, caspase-6 death domain-FK506 fusion protein, caspase-7 death domain-FK506 fusion protein, caspase-8 death domain-FK506 fusion protein, caspase-9 death domain-FK506 fusion protein, caspase-10 death domain-FK506 fusion protein, CD20, novel EC domains, and novel EC domains fused to chemical dimerization domain and intracellular death domain.

In another embodiment, the small molecule is converted into a cytotoxic agent that induces apoptosis in the genetically modified cells. In a related embodiment, the small molecule allows the multimerization of polypeptides that induce apoptosis upon multimerization. In a certain embodiment, the at least one cell suicide transgene encodes an RNAi molecule directed against a gene encoding a polypeptide that is essential for cell survival.

In another certain embodiment, the gene that encodes a polypeptide that is essential for cell survival is selected from the group consisting of: survivin, IAP, and Bcl2.

In a particular embodiment, the one or more cytotoxic agents are selected from the group consisting of: chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine,

trietylenephosphoramide, triethiylenethiophosphoramide and

novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;

nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics {e.g., calicheamicin, especially calicheamicin gammal I and calicheamicin omegaH (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,

cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,

doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,

methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as

ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;

pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;

(DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); aptamers, described for example in U.S. Patent No. 6,344,321 , which is herein incorporated by reference in its entirety; anti HGF monoclonal antibodies (e.g., AV299 from Aveo, AMG102, from Amgen); truncated c-Met variants {e.g., CGEN241 from Compugen); protein kinase inhibitors that block c-Met induced pathways {e.g., ARQ197 from Arqule, XL880 from Exelexis, SGX523 from SGX Pharmaceuticals, MP470 from Supergen, PF2341066 from Pfizer); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor {e.g., LURTOTECAN®); rmRH {e.g., ABARELIX®); lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); COX-2 inhibitors such as celecoxib (CELEBREX®; 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1 H-pyrazol- 1 -yl) benzenesulfonamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In one embodiment, each of the genetically modified cells comprises at least one factor transgene that encodes a dedifferentiation factor, a differentiation factor, a growth factor, a cytokine, or a hormone.

In another embodiment, an implant comprises at least one dedifferentiation factor polypeptide, differentiation factor polypeptide, growth factor polypeptide, cytokine, or hormone.

In a related embodiment, an implant comprises or more dedifferentiation agents, differentiation agents, or agents that increase cell growth and/or proliferation.

In a particular embodiment, the factor transgene encodes a growth factor or cytokine selected from the group consisting of: brain derived neurotropihic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (BMP-2), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 -CCL28, CXCL1 -CXCL17, XCL1 , XCL2, CX3CL1 , vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF), FAS-ligand, fibroblast growth factor 1 (FGF-1 ), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 (FGF-10), Flt-3, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM-CSF), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN-b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1 ), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 1 1 (IL-1 1 ), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 19 (IL-19), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derived growth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF- CC), platelet derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell derived factor 1 (SDF-1 ), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), Wnt1 , Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wntl Oa, Wntl Ob, Wnt1 1 , Wnt14, Wnt15, or Wnt16, Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

In a certain embodiment, the biocompatible matrix comprises one or more components selected from the group consisting of: a polymer, a biologically inert particle, a polypeptide, an oligosaccharide, a lipid, a

polynucleotide, or a small molecule or any combination thereof

In a certain particular embodiment, the polymer comprises one or more of polylactic acid, polyglycolic acid, polycaprolactone, polylactic acid- polyglycolic acid copolymer, polylactic acid-polycaprolactone copolymer, and polyglycolic acid-polycaprolactone copolymer. In one embodiment, the biologically inert particle is selected from the group consisting of: a magnetic particle, an agarose particle, and a plastic particle.

In another embodiment, the polypeptide is selected from the group consisting of: an antibody or antigen binding fragment thereof, a peptidomimetic, a lipoprotein, a ligand, a lectin, an Fc domain, and a cell- surface receptor or an extracellular fragment thereof.

In yet another embodiment, the polynucleotide is selected from the group consisting of: an aptamer, a single-stranded RNA, a double-stranded RNA, a hypomethylated single-stranded DNA molecule, and synthetic analogs thereof.

In a further embodiment, the small molecule is selected from the group consisting of: folate, biotin, digoxigenin, and dinitrophenyl.

In various other embodiment, the present invention provides, a composition comprising: one or more cytotoxic agents that target at least one population of susceptible cells; a plurality of genetically modified cells comprising, at least one resistance transgene that provides resistance to the one or more cytotoxic agents and at least one inducible cell suicide transgene; and a pharmaceutically acceptable cell culture medium. In particular embodiments, the cells are capable of being directed to a site in vivo, as described elsewhere herein,

In a particular embodiment, the plurality of genetically modified cells comprises a somatic cell. In certain embodiments, the plurality of genetically modified cells is isolated from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, hematopoietic cells, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, and kidney.

In certain other embodiments, the plurality of genetically modified cells is mammalian.

In certain particular embodiments, the plurality of genetically modified cells is human.

In one embodiment, a composition comprises a plurality of non- genetically modified cells. In a particular embodiment, the plurality of non- genetically modified cells is selected from the group consisting of: isolated adult stem cells, isolated embryonic stem cells, induced pluripotent stem cells, and cells differentiated therefrom.

In particualr embodiment, the at least one resistance transgene that provides resistance to one or more cytotoxic agents encodes a polypeptide selected from the group consisting of: ABCG2, BCRP, LRP, MRP1 , MRP2, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S- transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2, thrombopoietin receptor F36Vmpl, IRDS (interferons), HREP450R, SIRT1 , and variants, and functional fragments thereof.

In a particular related embodiment, the at least one inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: caspase-3, caspase-9, apoptosis inducing factor, BAD, and BIM.

In another particular embodiment, the at least one cell inducible cell suicide transgene encodes a polypeptide selected from the group

consisting of: thymidine kinase, HSVTK39, carboxylesterase ,

carboxypeptidase G2, cytochrome P450, cytosine deaminase, nitroreductase 1 , FAS-FK506 binding protein (FKBP) fusion protein, FADD-FK506 fusion protein, caspase-3 death domain-FK506 fusion protein, caspase-6 death domain-FK506 fusion protein, caspase-7 death domain-FK506 fusion protein, caspase-8 death domain-FK506 fusion protein, caspase-9 death domain-FK506 fusion protein, caspase-10 death domain-FK506 fusion protein, CD20, novel EC domains, and novel EC domains fused to chemical dimerization domain and intracellular death domain.

In a further embodiment, apoptosis is induced by contacting the plurality of genetically modified cells with a small molecule. In a further related embodiment, the small molecule is converted into a cytotoxic agent that induces apoptosis in the genetically modified cells.

In a certain embodiment, the small molecule allows the multimerization of polypeptides that induce apoptosis upon multimerization.

In one embodiment, the at least one cell suicide transgene encodes an RNAi molecule directed against a gene encoding a polypeptide that is essential for cell survival.

In another embodiment, the gene that encodes a polypeptide that is essential for cell survival is selected from the group consisting of: survivin, IAP, and Bcl2.

In yet another embodiment, the plurality of genetically modified cells is capable of being directed to a site in vivo, wherein the site is selected from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, or kidney.

trietylenephosphoramide, triethiylenethiophosphoramide and

lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-1 1 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocannptothecin); bryostatin;

nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics {e.g., calicheamicin, especially calicheamicin gammal I and calicheamicin omegaH (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin

pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;

toremifene (FARESTON®); anti-progesterones; estrogen receptor down- regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX®); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON® and ELIGARD®), goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate (MEGASE®), exemestane (AROMAS IN®), formestanie, fadrozole, vorozole (RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate

In another particular embodiment, each of the genetically modified cells comprises at least one factor transgene that encodes a dedifferentiation factor, a differentiation factor, a growth factor, a cytokine, or a hormone. In a certain embodiment, at least one dedifferentiation factor polypeptide, differentiation factor polypeptide, growth factor polypeptide, cytokine, or hormone.

In an additional embodiment, one or more dedifferentiation agents, differentiation agents, or agents that increase cell growth and/or proliferation.

In one embodiment, the factor transgene encodes a growth factor or cytokine selected from the group consisting of: brain derived neurotropihic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (BMP-2), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 -CCL28, CXCL1 -CXCL17, XCL1 , XCL2, CX3CL1 , vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF), FAS-ligand, fibroblast growth factor 1 (FGF-1 ), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 (FGF-10), Flt-3, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM-CSF), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN-b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1 ), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 1 1 (IL-1 1 ), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 19 (IL-19), macrophage colony- stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derived growth factor BB (PDGF- BB), platelet derived growth factor CC (PDGF-CC), platelet derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell derived factor 1 (SDF-1 ), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), Wnt1 , Wnt2,

Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt1 1 , Wnt14, Wnt15, or Wnt16, Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

In particular embodiments, expression of one or more transgenes is inducible.

In other particular embodiments, expression of one or more transgenes is constitutive. DETAILED DESCRIPTION

A. Overview

Cell-based compositions can be used in a variety of ways, including in vitro diagnostics, research, and therapy, which further includes the prevention, treatment, cure, amelioration, or mitigation of disease or injuries in mammals by the administration of autologous, allogeneic or xenogeneic cells.

Cell-based compositions are often manipulated or altered in vivo and/or ex vivo.

The goal of using cell-based compositions, overlapping with that of regenerative medicine, is to repair, replace or restore damaged tissues or organs. However, one major obstacle has been the inability those in the art to provide cell-based compositions with enhanced safety and efficacy for use in regenerative therapy.

Cell-based compositions of the present invention are directed, in part, to restoring health and treating disease by modulating cell fate with elevated safety and efficacy compared to methods presently available in the art.

Using the strategies provided by the present invention, cell-based therapy, optionally in combination with gene therapy, can also be used to safely and efficiently accomplish the repair, regeneration, restoration, or replacement of any particular cell, tissue, or organ.

Without wishing to be bound by any particular theory, the present invention contemplates, in part, to provide safer and more efficient cell-based compositions to modulate developmental signaling pathways that are associated with stem cell fate and/or that play a role in regenerative therapy in order to activate and modulate stem cells in the body to repair, regenerate, restore, or replace of any particular cell, tissue, or organ. Thus, the cell-based compositions of the present invention have been engineered with features that prevent and/or reduce many of the risks associated with conventional cell- based regenerative therapies.

The present invention further contemplates, in part, to provide personalized cell-based compositions that safely, efficiently, and reproducibly dedifferentiate and/or differentiate cells into specific types, including, but not limited to, inducing dedifferentiation and/or differentiation of adult stem cells in the body to treat diseases, disorders, or conditions requiring cell-based therapies.

For example, particular cell-based compositions of the present invention can be used to increase the engraftment, maintenance, and/or proliferation of stem cells to treat, ameliorate, and/or prevent various hematological diseases and hematological malignancies. When the desired level of therapy has been provided, then the cell-based compositions may be eliminated from the individual using the compositions and methods of the present invention.

Particular cell-based compositions of the present invention can also be used to safely promote, increase, and/or enhance the regeneration and restore the function of particular tissues in vivo. For example, cell-based compositions of the present invention that have enhanced safety features can restore the integrity of a damaged skin tissue in individuals suffering from traumatic injuries to the skin, including, but not limited to gashes, cuts, and burns. By way of another non-limiting example, cell-based compositions of the present invention can functionally restore the glial and/or neural cells of a damaged central or peripheral nervous system. Other examples for using cell- based compositions of the present invention, include but are not limited to, the functional restoration of damaged cardiac muscle and/or promoting angiogenesis in a damaged cardiac tissue or, the functional restoration of damaged bone or cartilage.

Particular cell-based compositions of the present invention can also be used to treat cancer patients by restoring hematopoietic cell populations and/or forcing cancer stem cells to differentiate and thus, lose the ability to proliferate and form metastases.

Other particular cell-based compositions of the present invention can also be used to increase the proliferation and/or differentiation of in vivo stem cells to increase or restore the function of a tissue damaged by

degenerative pathology. Exemplary degenerative pathologies include, but are not limited to, blindness, deafness, neurodegenerative diseases, diabetes, and coronary diseases.

Particular aspects of the present invention provide cell-based compositions that have enhanced safety measures, enhanced fail-safe measures, enhanced drug resistance and/or enhanced resistance to cytotoxic compounds.

Without wishing to be bound to any particular theory, the present invention contemplates, in part, that the safety and efficacy of cell-based therapies can be increased by precisely directing a therapeutic cell to an appropriate target site in vivo, inducing the cell to provide therapy, and subsequently, removing (e.g., by inducing self-elimination, apoptosis, programmed cell death) the therapeutic cell from the site once the desired level of therapy has been achieved. For example, a stem cell genetically modified to produce a growth factor or cytokine can be administered to an individual and directed to a particular site using methods known in the art, particularly those methods described in U.S. Provisional Patent Application No. 61/241 ,71 1 , filed on September 1 1 , 2009, and entitled "Cell-based compositions and uses thereof," which is herein incorporated by reference in its entirety. Once the desired amount of therapy has been achieved, the cells can be induced to undergo apoptosis; thus nullifying the risk that the cell-based therapy will pose any safety concerns following the course of therapy. Thus, in various embodiments, the present invention provides, in part, a method for providing a cell-based therapy comprising: the

administration of a composition comprising a plurality of genetically modified cells, wherein the genetically modified cells are directed to a site in vivo and one or more cytotoxic agents and wherein the genetically modified cells are resistant to the cytotoxic agent; allowing the genetically modified cells to remain at the in vivo site under conditions and for a time sufficient to provide an amount of therapy to the individual; and/or inducing programmed cell death in the genetically modified cells, thereby removing the genetically modified cells from the individual, whereby removal of the genetically modified cells increases the safety of and/or decreases the risks associated with the cell-based therapy.

In additional embodiments, the present invention provides, in part, a composition comprising: one or more cytotoxic agents that target at least one population of susceptible cells; a plurality of genetically modified cells that have at least one resistance transgene that provides resistance to the one or more cytotoxic agents and at least one inducible cell suicide transgene; and a pharmaceutically acceptable cell culture medium.

In further embodiments, the present invention provides, in part, a composition comprising: a plurality of genetically modified cells that have at least one inducible cell suicide transgene; and a pharmaceutically acceptable cell culture medium.

In various other embodiments, the present invention provides, in part, an implant comprising: one or more cytotoxic agents; a cell-based composition comprising a plurality of genetically modified cells, wherein the genetically modified cells comprise one or more resistance transgenes that provide resistance to the one or more cytotoxic agents and one or more inducible cell suicide transgenes; a biocompatible matrix; and a

pharmaceutically acceptable cell culture medium.

In various other embodiments, the present invention provides, in part, a kit comprising: one or more cytotoxic agents; a cell-based composition comprising a plurality of genetically modified cells, wherein the genetically modified cells comprise one or more resistance transgenes that provide resistance to the one or more cytotoxic agents and/or one or more inducible cell suicide transgenes and/or a biocompatible matrix and/or a pharmaceutically acceptable cell culture medium.

In various other embodiments, the present invention provides, in part, a composition comprising: one or more cytotoxic agents that target at least one population of susceptible cells; a plurality of genetically modified cells that have at least one resistance transgene that provides resistance to the one or more cytotoxic agents and at least one inducible cell suicide switch; and a pharmaceutically acceptable cell culture medium.

B. Cells

In various embodiments, compositions and methods of the present invention provide cell-based compositions comprising one or a plurality of cells that can be directed to a particular site in vivo to effect therapy. As used herein, the term "plurality" refers means more than one. "A plurality of cells" is often used interchangeably with the term "population of cells". A plurality of cells refers to at least 2 , 3, 4 , 5, 6 , 7, 8, 9, 10, 100, 10³, 10⁴, 10⁵ , 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹², 10¹⁵ or more total cells or cells per unit area or cells per unit volume. Exemplary units of area, include, without limitation, μιτι², mm², cm², and m². Exemplary units of volume include, without limitation, pL, μΙ_, ml_, and L. The skilled artisan will appreciate that any intervening number of cells at any particular concentration {e.g., per unit area or per unit volume) can be used in particular embodiments of the present invention.

As used herein, the term "cell-based composition" refers to a single cell, a plurality, or a population of cells. The cell-based composition comprises any number and/or combination of homogenous or heterogeneous cell types, as described elsewhere herein. In particular embodiments, cell- based compositions of the present invention comprise isolated primary cells or cell lines, either of which can comprise one or more genetic modifications and/or transgenes. In particular embodiments a cell-based composition comprises at least one plurality of: genetically modified cells; non-genetically modified cells; or combinations thereof.

Cells can be obtained and isolated from a reptilian species, an avian species, a species of fish, or any mammalian species. Exemplary mammals include, but are not limited to, humans and non-human primates such as baboons, gorillas, chimpanzees, rhesus macaques and other non-human primates, and also include equine, bovine, sheep (ovine), goat (caprine), porcine, canine, feline, chicken, rat, and mouse (murine) species. In particular embodiments, the mammal is a human.

In particular embodiments, an individual can be administered or treated with syngeneic, isogeneic, allogenic, or xenogenic cells.

In various embodiments, cell-based compositions comprise one or a plurality of stem cells, progenitor cells, differentiated cells, or any combination thereof. The cells can be adult or embryonic in origin. In particular

embodiments, cell-based compositions comprise at least one cell selected from the group consisting of: an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell. In particular embodiments, a cell- based composition comprises one or more induced pluripotent stem cells, and/or cells differentiated therefrom. In another embodiment, a cell-based composition comprises one or more embryonic and/ or adult stem cells, and/or cells differentiated therefrom.

Stem cells, progenitor cells, and/or differentiated cells can be isolated from any desired tissue or organ using methods known to those having ordinary skill in the art. For example, cells can be isolated from the pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose tissue, lung, retina, cornea, and kidney.

As used herein, the term "stem cell" refers to a cell that can self- renew and that has sufficient potency to differentiate into more specialized cell types. For example, an embryonic stem cell (ESC) has the capacity to self- renew indefinitely and may differentiate into any cell type of the embryo proper. As used herein, the term "progenitor cell" refers to a cell with a limited capacity for self-renewal that spans several rounds of cell division before terminally differentiating. As used herein, the term "differentiated cell" refers to a cell that normally does not have the capacity to self-renew. Methods of differentiating stem/progenitor cells are known in the art.

As used herein, the term "self-renew" refers to the ability to go through numerous cycles of cell division while maintaining an undifferentiated state. As used herein, the term "potency" refers to the sum of all developmental options accessible to the cell (i.e., the developmental potency). One having skill in the art would recognize that cell potency is a continuum, ranging from the totipotent stem cell to the terminally differentiated cell. The continuum of cell potency includes, but is not limited to, totipotent cells, pluripotent cells, multipotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells. In the strictest sense, stem cells are either totipotent or pluripotent; thus, being able to give rise to any mature cell type. However, multipotent, oligopotent or unipotent progenitor cells are sometimes referred to as lineage restricted stem cells (e.g., mesenchymal stem cells, adipose tissue derived stem cells, etc.) and/or progenitor cells.

It would also be clear to one having skill in the art that potency can be partially or completely altered to any point along the developmental lineage of a cell (i.e., from totipotent to terminally differentiated cell), regardless of cell lineage. One having skill in the art would further recognize that terminally differentiated somatic cells may be reprogrammed or dedifferentiated into totipotent, pluripotent, and multipotent cells (collectively referred to as reprogrammed cells); thus, providing another source of cells suitable for use as a cell-based composition in various embodiments of the present invention.

Cells reprogrammed to a pluripotent state are referred to herein as induced pluripotent stem cells (iPSCs). Methods of somatic cell reprogramming are known in the art and include each and every reprogramming method disclosed in the U.S. Provisional Patent Application entitled "Reprogramming

Compositions and Methods of Using the Same", serial number 61 /241 ,647, which is herein incorporated by reference in its entirety. Particularly, Section I, entitled Overview of Somatic Cell Reprogramming"; Section II, entitled of "Stem Cells of Different Origins"; and Section IX, entitled "Repressors and Activators" of 61/241 ,647 are herein incorporated by reference in their entireties.

As used herein, the term "totipotent" refers to the ability of a cell to form all cell lineages of an organism. For example, in mammals, only the zygote and the first cleavage stage blastomeres are totipotent. As used herein, the term "pluripotent" refers to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper). For example, embryonic stem cells are a type of pluripotent stem cell that are able to form cells from each of the three germs layers, the ectoderm, the mesoderm, and the endoderm. As used herein, the term "multipotent" refers to the ability of an adult stem cell to form multiple cell types of one lineage. For example, hematopoietic stem cells are capable of forming all cells of the blood cell lineage, e.g., lymphoid and myeloid cells. As used herein, the term "oligopotent" refers to the ability of an adult stem cell to differentiate into only a few different cell types. For example, lymphoid or myeloid stem cells are capable of forming cells of either the lymphoid or myeloid lineages, respectively. As used herein, the term

"unipotent" means the ability of a cell to form a single cell type. For example, spermatogonia! stem cells are only capable of forming sperm cells.

According to the methods and compositions of the present invention a cell-based composition or homing cell may comprise a primary cell isolated from an in vivo tissue or a cell derived from an in vitro cell line. One of ordinary skill in the art is familiar with methods for isolating and culturing the different types of cells described herein (e.g., Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001 ); Hematopoietic Stem Cell Protocols (Methods in

Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008)).

Cells may be cultured in growth-promoting conditions, which can include any set of conditions (temperature, atmosphere, growth medium composition, humidity, degree of agitation, etc.) under which cells normally proliferate. None of these conditions are critical. The temperature should be near that of normal human body temperature (i.e., about 37°C), but can be any temperature at which cells can proliferate (e.g., 30°C to 43°C). Cells can be grown in an air atmosphere, or an air atmosphere supplemented with 5% CO2, for example. The growth medium can be any liquid medium which contains nutrients and factors sufficient to support proliferation of cells. Such media contain, for example, a carbon source (e.g., glucose) and minimal essential nutrients, and preferably contain one or more of a mammalian serum (e.g., fetal calf serum), an antibiotic (e.g., penicillin or streptomycin), and L-glutamine (i.e., to improve amino acid supply for protein biosynthesis).

Mammalian serum can be used at a concentration of 1 % to 20%, by volume, of the total growth medium. The serum is preferably pre-screened to ensure that it supports vigorous growth of cells; some lots, even lots provided from the same supplier, do not support vigorous growth of cells. Alternatively, the mammalian serum can be replaced with one or more growth factors (e.g., fibroblast growth factor, platelet derived growth factor, insulin growth factor, or endothelial growth factor). The growth medium can, for example, be Minimal Essential Medium-alpha without deoxyribonucleotides or ribonucleotides, supplemented with fetal calf serum, antibiotics, and L-glutamine; Dulbecco's minimal essential medium; and others well known to one of ordinary skill in the art. The growth medium is preferably replaced one or more times (e.g., every 3 or 4 days) during culture of the cells.

In various illustrative embodiments, both cell-based compositions comprise one or more transgenes as described elsewhere herein. Methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA are described for cells in general, for example, in Sambrook et al. (2001 , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor

Laboratory, New York), and in Ausubel et al. (2007, Current Protocols in

Molecular Biology, John Wiley & Sons, New York), and elsewhere herein.

Cell-based compositions of the present invention are often administered, in an effective amount. As used herein, the term "effective amount" includes an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

A "therapeutically effective amount" of a cell-based composition of the invention, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cell-based composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cell-based composition are outweighed by the therapeutically beneficial effects.

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired

prophylactic result. Typically, but not necessarily, a prophylactic dose is used in subjects prior to or at an earlier stage of disease; thus, the prophylactically effective amount is less than the therapeutically effective amount.

In one illustrative embodiment, the effective amount of cells in a cell-based composition provided to a subject is less than 1 x 10¹² cells per 100 kg, less than 1 x 10¹¹ cells per 100 kg, less than 1 x 10¹⁰ cells per 100 kg, less than 1 x 10⁹ cells per 100 kg, less than 1 x 10⁸ cells per 100 kg, less than 1 x 10⁷ cells per 100 kg, less than 5 x 10⁶ cells per 100 kg, less than 4 x 10⁶ cells per 100 kg, less than 3 x 10⁶ cells per 100 kg, less than 2 x 10⁶ cells per 100 kg, less than 1 x 10⁶ cells per 100 kg, less than 5 x 10⁵ cells per 100 kg, less than 4 x 10⁵ cells per 100 kg, less than 3 x 10⁵ cells per 100 kg, less than 2 x 10⁵ cells per 100 kg, less than 1 x 10⁵ cells per 100 kg, less than 5 x 10⁴ cells per 100 kg, or less than 1 x 10⁴ cells per 100 kg.

In another illustrative embodiment, the effective amount of cells in a cell-based composition provided to a subject is about 1 x 10¹² cells per 100 kg, about 1 x 10¹¹ cells per 100 kg, about 1 x 10¹⁰ cells per 100 kg, about 1 x 10⁹ cells per 100 kg, about 1 x 10⁸ cells per 100 kg, about 1 x 10⁷ cells per 100 kg, about 5 x 10⁶ cells per 100 kg, about 4 x 10⁶ cells per 100 kg, about 3 x 10⁶ cells per 100 kg, about 2 x 10⁶ cells per 100 kg, about 1 x 10⁶ cells per 100 kg, about 5 x 10⁵ cells per 100 kg, about 4 x 10⁵ cells per 100 kg, about 3 x 10⁵ cells per 100 kg, about 2 x 10⁵ cells per 100 kg, about 1 x 10⁵ cells per 100 kg, about 5 x 10⁴ cells per 100 kg, or about 1 x 10⁴ cells per 100 kg.

In another illustrative embodiment, the effective amount of cells in a cell-based composition provided to a subject is from about 1 x 10¹ cells per 100 kg to about 1 x 10¹² cells per 100 kg, from about 1 x 10² cells per 100 kg to about 1 x 10^{1 1} cells per 100 kg, from about 1 x 10³ cells per 100 kg to about 1 x 10¹⁰ cells per 100 kg, from about 1 x 10⁴ cells per 100 kg to about 1 x 10⁹ cells per 100 kg, from about 1 x 10⁵ cells per 100 kg to about 1 x 10⁸ cells per 100 kg, from about 1 x 10⁶ cells per 100 kg to about 1 x 10⁷ cells per 100 kg, or any intervening ranges of cells per 100 kg.

Cell and/or cell-based compositions are administered to an individual by various routes known to those of ordinary skill in the art. As used herein, the term "administration" or "administering" is used throughout the specification to describe the process by which cells and/or cell-based compositions are delivered to an individual for therapeutic purposes.

Administration of the compositions of the present invention can be

accomplished in a number of ways, including, but not limited to, parenteral (such term referring to intravenous and intraarterial as well as other appropriate parenteral routes), intrathecal, intraventricular, intraparenchymal (including into the spinal cord, brainstem or motor cortex), intracisternal, intracranial, intrastriatal, and intranigral, among others, which allow the stromal cells used in the methods of the present invention to ultimately migrate to the in vivo target site wherein therapy is desired.

In particular embodiments, administration can be modified upon the disease or condition treated and may preferably be via a parenteral route, for example, intravenously, or intraarterially, or by direct administration into the tissue or organ wherein therapy is desired.

C. Resistance Mechanisms

Various treatment plans include treating an individual with a drug, agent, or compound that is cytotoxic to a targeted population of cells. For example, a targeted population can be a rapidly proliferating population of cells and/or a population of cells that displays particular cell surface antigen that enables a therapeutic to target the cell. Unfortunately, while many abherrent and/or undesirable cells possess the characteristic used to target the cells, non- target cells are often unintentionally targeted because they may also be rapidly proliferating cells or cells that display the particular cell-surface antigen being targeted. For example, chemotherapeutics often target rapidly proliferating cancer cells, but in addition, they also target normal rapidly proliferating cells of the hematopoietic system, such as hematopoietic stem cells. This situation results in a treated individual that is severely immunocomprised and thus, vulnerable to secondary complications, such as systemic infection.

The compositions and methods of the present invention, provide in part, cell-based compositions that are administered to an individual, wherein the compositions comprise one or more cytotoxic agents and at least one plurality of genetically modified cells, wherein the genetically modified cells are resistant to the one or more cytotoxic agents.

Exemplary cytotoxic agents include, but are not limited to chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan,

improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins

(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol

(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-1 1 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1 - TM1 ); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfanide, mechlorethamine, mechlorethamine oxide

hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammal I and calicheamicin omegaH (see, e.g., Agnew, Chem Intl. Ed. Engl., 33: 183- 186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,

chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCI liposome injection (DOXIL®) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine

(GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); folic acid analogues such as denopte n,

pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;

ABARELIX®); lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also known as GW572016); COX-2 inhibitors such as celecoxib (CELEBREX®; 4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1 H-pyrazol- 1 -yl) benzenesulfonamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Cells can be genetically modified to express 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more resistance genes. As used herein, the terms "resistance gene" and "resistance transgene" refer to genes that encode proteins that provide an increase in resistance to a treatment, drug, or compound. Exemplary resistance genes include, but are not limited to: ABCG2, BCRP, LRP, MRP1 , MRP2, MRP3, MRP4, MRP5, MRP6, MRP8, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S-transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2, thrombopoietin receptor F36Vmpl, and variants and functional fragments thereof.

As noted above, an exemplary group of resistance genes include ABC transporter proteins. ABC transporters are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out certain biological processes including translocation of various substrates across membranes and non-transport-related processes such as translation of RNA and DNA repair. They transport a wide variety of substrates across extra- and intracellular membranes, including metabolic products, lipids and sterols, and drugs. Proteins are classified as ABC transporters based on the sequence and organization of their ATP-binding cassette (ABC) domain(s). ABC transporters are involved in tumor resistance, cystic fibrosis, bacterial multidrug resistance, and a range of other inherited human diseases.

Exemplary ABC transporter genes that encode proteins that provide an increase in resistance to a treatment, drug or compound, but are not limited to: ATP-binding cassette, sub-family B (MDR/TAP), member 1 (also known as ABCB1 , PGY1 , or MDR1 ); multidrug resistance protein 1 (MRP1 or ABCC1 ); multidrug resistance protein 2 (MRP2 or ABCC2); multidrug

resistance protein 3 (MRP3 or ABCC3); multidrug resistance protein 4 (MRP4 or ABCC4); multidrug resistance protein 6 (MRP6 or ABCC6); multidrug resistance protein 5 (MRP5 or ABCC5); multidrug resistance protein 8 (MRP8 or ABCC8);and ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2). Exemplary treatments, drugs, and/or compounds for which MDR1 provides resistance to include, but are not limited to colchicine, tacrolimus, quinidine, etoposide, doxorubicin, vinblastine, vincristine, actinomycin D, taxol, paclitaxil, VP16, verapamil, PSC833, GG918, adriamycin, digoxin, V-104, pluronic L61 saquinivir, daunorubicin, cyclosporin A, and V-104, among others.

Exemplary treatments, drugs, and/or compounds for which MRP1 provides resistance to include, but are not limited to, colchicine, etoposide, and rhodamine, among others.

Exemplary treatments, drugs, and/or compounds for which MRP2 provides resistance to include, but are not limited to vinblastine, sulfinpyrazone, anthracin, vincristine, etoposide, campothecin, methotrexate, and platinum- based compounds, among others.

Exemplary treatments, drugs, and/or compounds for which MRP3 provides resistance to include, but are not limited to etoposide, methotrexate, and cyclic nucleotides, among others.

Exemplary treatments, drugs, and/or compounds for which MRP4 provides resistance to include, but are not limited to nucleoside

monophosphates, etoposide, methotrexate, 6-mecaptopurine (6-MP), paramethoxyethylamphetamine (PMEA), and cyclic nucleotides, among others. Exemplary treatments, drugs, and/or compounds for which MRP5 provides resistance to include, but are not limited to mitoxantrone, topotecan, doxorubicin, fumitremorgin C, GF120918, 6-MP, PMEA, and cyclic nucleotides, among others.

Exemplary treatments, drugs, and/or compounds for which MRP6 provides resistance to include, but are not limited to anthracin, etoposide, and platinum-based compounds, among others.

Exemplary treatments, drugs, and/or compounds for which MRP8 provides resistance to include, but are not limited to 5-fluorouracil (5-FU), 2'-3'- dideoxycytidine (ddC), PMEA, and cyclic nucleotides, among others. Exemplary treatments, drugs, and/or compounds for which

ABCG2 provides resistance to include, but are not limited to daunorubicin, vincristine, etoposide, campothecin 1 1 , CPT-1 1 , and rhodamine, among others.

The O-6-methylguanine-DNA methyltransferase (MGMT) gene encodes a protein that provides an increase in resistance to various treatments, drugs and compounds. Exemplary treatments, drugs, and/or compounds for which MGMT provides resistance to include, but are not limited to: alkylating agents, such as, for example, carmustine, 3-[(4-amino-2-methyl-pyrimidin-5- yl)methyl]-1 -(2-chloroethyl)-1 -nitroso-urea (ACNU), temozolomide, dacarbazine, procarbazine; and O6-benzylguanine (O6BG), among others.

Aldehyde dehydrogenases also provide an increase in resistance to various treatments, drugs and compounds. Exemplary treatments, drugs, and/or compounds for which aldehyde dehydrogenases provides resistance to include, but are not limited to oxazaphosphorines (e.g., cyclophophamide), among others.

Glutathione S-transferase also provides an increase in resistance to various treatments, drugs and compounds. Exemplary treatments, drugs, and/or compounds for which glutathione S-transferase provides resistance to include, but are not limited to alkylating agents, anthracins, doxorubicin, cisplatin, cyclophosphamide, and melphalan, among others.

Additional genes that encode for proteins that provide resistance to various treatments, drugs and compounds include, but are not limited to: mutant dihydrofolate reductase, which provides resistance against

methotrexate, trimetrexate, and the like; thymidylate synthase which provides resistance against 5-Fluorouracil, and the like; and cytosine deaminase which provides resistance against Ara-c, gemcitibine, and the like.

Furthermore, mechanisms to increase the resistance of a cell to a cytotoxic treatment, drug, or compound includes expression of one or more genes that antagonize or inhibit apoptosis in a cell . Exemplary genes that antagonize apoptosis include, but are not limited to survivan, inhibitor of apoptosis protein, and Bcl-2.

Other mechanisms to increase resistance of a cell to a cytotoxic treatment, drug, or compound includes small molecule based dimerization systems that lead to drug-dependent increases in cell growth. For example, a patient receiving bone marrow ablative therapy can be administered a population of hematopoietic stem cells comprising a thrombopoietin receptor mutant (e.g.,F36Vnnpl), wherein activation of the receptor through

administration of a dimerizing drug, such as, for example, AP20187, produces reversible, drug-dependent rises in genetically modified hematopoietic stem cells. Thus, the patient's hematopoietic system is reconstituted, with minimal side effects.

One of skill in the art would appreciate that the foregoing description of resistance genes and mechanisms is not exhaustive and is intended to provide an exemplary set of tools that the artisan can use in practicing particular embodiments of the invention.

D. Cell Suicide Mechanisms

Cell-based therapies that are currently implemented in the art suffer from a number of safety issues, as discussed elsewhere herein. The present invention contemplates, in particular embodiments, that once a cell- based therapy has been provided to a patient for a time sufficient to elicit therapy it is preferred or often desirable to remove or eliminate the therapeutic cell following the therapy, in order to remove the risk of causing malignancy or unforeseen complications, including, but not limited to rejection of the cells by the host immune system.

Thus, in particular embodiments, the present invention provides a method for providing a cell-based therapy that comprises administration of a plurality of genetically modified cells to an individual, wherein the cells are directed to a site in vivo; and allowed to remain at the site under conditions and for a time sufficient to provide an amount of therapy to the individual. Once therapy has been achieved, apoptosis or cell suicide is induced in the genetically modified cells. In preferred embodiments, the apoptosis or cell suicide program is specific to the genetically modified cells and does not target non-target cells to undergo programmed cell death. In various embodiments, the cell specific apoptosis is accomplished by genetically modifying cells to controllably express at least one cell suicide transgene.

In other particular embodiments, the present invention provides a method for providing a cell-based therapy that comprises administration, to an individual, of a plurality cells that have been genetically modified to express one or more resistance transgenes, wherein the cells are directed to a site in vivo; and allowed to remain at the site under conditions and for a time sufficient to provide an amount of therapy to the individual. Once therapy has been achieved, apoptosis or cell suicide is induced in the genetically modified cells (i.e., the genetically modified cells comprising the one or more resistance transgenes). Thus, in one aspect, the present invention provides a population of genetically modified cells, wherein each cell has been genetically modified to express one or more resistance transgenes and to controllably express at least one cell suicide transgene.

In preferred embodiments, the apoptosis or cell suicide program is specific to the genetically modified cells and does not target non-target cells to undergo programmed cell death. In various embodiments, the cell specific apoptosis is accomplished by further genetic modification of the cells that express one or more resistance transgenes to controllably express at least one cell suicide transgene. The foregoing methods enhance the safety of the cell- based therapy.

Exemplary components of cell suicide transgene systems include, but are not limited to: pro-drug systems comprising expression of a factor that converts a non-toxic pro-drug into a toxic drug; RNAi systems that target cell survival genes; small molecule regulated cell death inducing proteins; systems in which a cell is genetically modified to be dependent upon a nutrient and where withdrawal of the nutrient induces apoptosis; and other systems designed to initiate programmed cell death in the cell-based compositions.

The use of prodrugs has been actively pursued to achieve very precise and direct effects at the "site of action," with minimal effect on the rest of the body (see, Stella and Himmelstein. Prodrugs and site-specific drug delivery. J Med Chem. 1980; 23: 1275-1282; Stella and Himmelstein. Critique of prodrugs and site specific delivery. In: Bundgaard H, ed. Optimization of Drug Delivery. Alfred Benzon Symposium 17. Copenhagen, Munksgaard;

1982:134-155).

In particular embodiments, the present invention provides a method of cellular therapy, comprising: directing one or a plurality of cells to a target site, each cell comprising at least one inducible transgene; allowing the cells to remain at the site under conditions and for a time sufficient to provide therapy; and inducing the expression of the transgene, thereby inducing apoptosis in the cells. In certain embodiments, the inducible transgene is an enzyme that converts a non-toxic prodrug into a toxic drug in the cell, thereby inducing apoptosis in the cell.

As used herein, the term "prodrug" refers to a pharmacologically inert substance that can be enzymatically or non-enzymatically converted in vivo to a pharmacologically active drug molecule. In preferred embodiments, the prodrug is non-toxic and the corresponding activated drug is cytotoxic.

Without wishing to be bound to any particular theory, it is contemplated that after a therapeutic cell has provided a sufficient amount of therapy, the risks of having the therapeutic cells remain at the in vivo site outweigh any further benefits that could be attained by allowing the cell to remain at the site; thus, in particular embodiments it is preferable to induce the therapeutic cells to undergo apoptosis once they have served their purpose. In particular embodiments, the therapeutic cells of the invention comprise an inducible transgene, that inducibly catalyzes the conversion of a specific non- toxic prodrug into a toxic drug and thereby induces apoptosis in the cell, in effect causing the therapeutic cells to commit suicide. One having ordinary skill in the art would appreciate that such a strategy is specifically targeted to the therapeutic cells and does not influence the viability of non-target cells that are adjacent or nearby.

Exemplary cell-specific or targeted prodrug systems that are suitable for inducing cell suicide in genetically modified cells in particular embodiments of the present invention include, but are not limited to: a herpes virus thymidine kinase transgene {e.g., HSVTK39) that catalyzes the

conversion of the non-toxic prodrugs acyclovir, ganciclovir, and the like, to the cytotoxic drugs acyclovir triphosphate, ganciclovir triphosphate, respectively; a carboxylesterase transgene that catalyzes the conversion of the non-toxic prodrug Irinotecan to the cytotoxic drug 7-ethyl-10-hydroxy-camptothecin; a carboxypeptidase G2 transgene that catalyzes the conversion of the non-toxic prodrug 4-[(2-Chloroethyl)(2-mesyloxyethyl)amino]benzoyl -L-glutamic acid (CMDA) to the cytotoxic drug 4-[(2-Chloroethyl)(2-mesyloxyethyl)amino]benzoic acid (CMBA); a cytochrome P450 transgene that catalyzes the conversion of the non-toxic prodrug cyclophospamide to the cytotoxic drug 4- hydroxycyclophosphamide, which degrades into phosphoramide mustard; cytosine deaminase that catalyzes the conversion of the non-toxic prodrug 5- Fluorocytosine to the cytotoxic drug 5-Fluoruracil; and nitroreductase 1 that catalyzes the conversion of the non-toxic prodrug 5-Aziridinyl-2,4- dinitrobenzamide (CB1954) to the cytotoxic drug 5-(Aziridin-1 -yl)-4- hydroxylamino-2-nitrobenzamide.

RNAi-based systems that target cell survival genes can also be used in a cell specific manner to induce cell suicide in the cell-based

compositions comprising genetically modified cells. For example, in particular embodiments, a cell suicide transgene comprising an RNAi construct is designed to inducibly express an RNAi molecule {e.g., siRNA, miRNA, shRNA) that is directed against a cell survival gene. Exemplary cell survival genes that can be targeted by the RNAi molecule include, but are not limited to surviving, inhibitor of apoptosis protein, and Bel. Exemplary small molecule based dimerization systems that are suitable for inducing cell suicide in genetically modified cells in particular embodiments of the present invention include, but are not limited to: a transgene that is designed to express a fusion polypeptide comprising a FAS, FADD, caspase-3, 6, 7, 8, 9, 12 (CARD domain), or 10 death domain fused to a chemical dimerization domain, e.g., FK506 binding protein (FKBP). Such apoptosis inducing fusion polypeptides can be activated to dimerize and/or induce apoptosis upon binding to a small molecule, such as, for example, FK506, rapamycin, cyclosporine, coumermycin, AP1510, AP1902, and

AP20187.

In particular embodiments, apoptosis inducing fusion polypeptides comprise a novel extracellular (EC) domain fused to an intracellular FAS, FADD, caspase-3, 6, 7, 8, 9, 12 (CARD domain), or 10 death domain. The EC domain is designed to recognize an engineered ligand that has no natural cognate receptor. Upon ligand binding to the EC domain, the fusion

polypeptides oligomerize, activate the apoptosis pathway via the death domain, and induce cell death.

In other particular embodiments, apoptosis inducing fusion polypeptides comprise a novel extracellular (EC) domain that recognizes an engineered ligand, and a FAS, FADD, caspase-3, 6, 7, 8, 9, 12 (CARD domain), or 10 death domain fused to a chemical dimerization domain, e.g., FK506 binding protein (FKBP). Upon binding to a small molecule, such as, for example, FK506, rapamycin, cyclosporine, coumermycin, AP1510, AP1902, and AP20187, the fusion polypeptides oligomerize, activate the apoptosis pathway via a death domain, and induce cell death.

In one embodiment, a cell is genetically modified so as to become dependent upon a nutrient that must be exogenously supplied in order to maintain cell viability. For example, an enzyme that catalyzes the synthesis of an essential nutrient can be "knocked-out" and the nutrient may instead, be supplied to the cell to ensure viability. However, once the nutrient is withdrawn, the cell initiates apoptosis. One of skill in the art would appreciate that the foregoing description of cell-suicide genes and mechanisms is not exhaustive and is intended to provide an exemplary set of tools that the artisan can use in practicing particular embodiments of the invention. E. Cell Fate Modulators

In various embodiments of the invention, genetically modified cells of the invention comprise at least one factor transgene that encodes a dedifferentiation factor, a differentiation factor, a growth factor or a cytokine.

As used herein, the term "factor transgene" refers to a transgene, as described elsewhere herein, wherein the transgene comprises a

polynucleotide that encodes for a polypeptide that modulates, affects, or is associated with cell dedifferentiation, cell differentiation, or cell proliferation. In particular embodiments, a factor transgene is provided that includes one or more factor transgenes. For example, in particular embodiments, a transgene comprises one or more polynucleotides encoding one or more factors. The factors can be separated by internal ribosomal entry sites (IRES) or by sequences encoding a foot and mouth virus self-cleaving peptide (2A peptide). In this manner, a single transgene can be used to express multiple protein factors of the invention in a single cell.

Exemplary dedifferentiation factors include, but are not limited to,

Oct-4, Nanog, Sox-2, cMyc, Klf-4, Lin-28, Stat-3, Tcf-3, hTERT, Stella, Rex-1 , UTF-1 , Dax-1 , Nac-1 , Sail 14, TDGD-1 , and Zfp-281 .

Exemplary differentiation factors, growth factor, and cytokines include, but are not limited to brain derived neurotropihic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (BMP-2), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 -CCL28, CXCL1 -CXCL17, XCL1 , XCL2, CX3CL1 , vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF), FAS-ligand, fibroblast growth factor 1 (FGF-1 ), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 (FGF-10), Flt-3, granulocyte colony- stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM- CSF), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN-b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1 ), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 3 (IL- 3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL- 7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 1 1 (IL-1 1 ), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL-17), interleukin 19 (IL-19), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP- 3b), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derived growth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF-CC), platelet derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell derived factor 1 (SDF-1 ), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), Wnt1 , Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt1 1 , Wnt14, Wnt15, or Wnt16, Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

In certain embodiments, a genetically modified cell comprises one or more factor transgenes that contribute to the therapeutic response of the individual being treated. Without wishing to be bound by any particular theory, when a cell-based composition of the invention is targeted to a site in vivo, a cell having one or more factor transgenes can increase the rate of therapy by providing factors that increase cell proliferation, differentiation, dedifferentiation, or any combination thereof. F. Polynucleotides

The present invention also provides isolated polynucleotides and polynucleotides encoding polypeptides (e.g., resistance polypeptides, cell- suicide polypeptides, and factor peptides) of the invention, as described elsewhere herein. Isolated polynucleotides of the present invention, include, but are not limited to, resistance transgenes, cell-suicide transgenes, and factor transgenes, as described elsewhere herein. Fusion polynucleotides that encode fusion polypeptides are also included in the present invention, as described elsewhere herein.

The terms "polynucleotide" and "polynucleotide" include

polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNAn or DNA, including, but mot limited to RNA and DNA analogs. Polynucleotides include, without limitations, single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-strand regions, hybrid molecules including DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the term "polynucleotide" refers to triple stranded regions of RNA or DNA or both RNA and DNA.

The term "polynucleotide" also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine, as well as others described elsewhere herein. A variety of modifications have been made to DNA and RNA. Thus, the term "polynucleotide" embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as chemical forms of DNA and RNA, and DNA and RNA characteristic of viruses and cells. The term "polynucleotide" further includes but is not limited to linear and end-closed molecules. "Polynucleotide" also embraces relatively short polynucleotides, often referred to as oligonucleotides. In various illustrative embodiments, the present invention provides isolated polynucleotides that encode polypeptides of the invention, including, but not limited to those polypeptides described elsewhere herein.

Polynucleotides can be synthesized using protocols known in the art as described in Caruthers et al., 1992, Methods in Enzymology 21 1 , 3-19; Thompson et al., International PCT Publication No. WO 99/54459; Wincott et al., 1995, Polynucleotides Res. 23, 2677-2684; Wincott et al., 1997, Methods Mol. Bio., 74, 59-68; Brennan et al., 1998, Biotechnol Bioeng., 61 , 33-45; and Brennan, U.S. Pat. No. 6,001 ,31 1 .

As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar.

Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1 ^' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and others (see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187)). There are several examples of modified polynucleotide bases known in the art as summarized by Limbach et al., 1994, Polynucleotides Res. 22, 2183-2196.

As used herein, the term "modified bases" means nucleotide bases other than adenine, guanine, cytosine, thymine, and uracil at 1 ^' position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic polynucleotide molecule and/or in the substrate-binding regions of the polynucleotide molecule.

As used herein, the term "nucleoside" means a heterocyclic nitrogenous base in N-glycosidic linkage with a sugar. Nucleosides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the 1 ^' position of a nucleoside sugar moiety. Nucleosides generally comprise a base and sugar group. The nucleosides can be unmodified or modified at the sugar, and/or base moiety, (also referred to interchangeably as nucleoside analogs, modified nucleosides, non-natural nucleosides, non-standard nucleosides and others ( see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187).

As used herein, the terms "DNA" and "deoxyribonucleotide" and "polydeoxyribonucleotide" refer to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding a polypeptide refers to a DNA segment that contains one or more coding sequences yet is substantially isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the terms "DNA" and "deoxyribonucleotide" and

"polydeoxyribonucleotide" are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.

As will be understood by those skilled in the art, the polynucleotide sequences of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides, and the like. Such segments may be naturally isolated, recombinant, or modified synthetically by the hand of man.

Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a polypeptide of the invention or a portion thereof) or may comprise a variant, or a biological functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the activity and/or three dimensional structure of the encoded polypeptide is not substantially altered relative to the unmodified polypeptide. The polynucleotides of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

Polynucleotides and fusions thereof may be prepared, manipulated and/or expressed using any of a variety of well established techniques known and available in the art. For example, polynucleotide sequences which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a polypeptide of the present invention. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode

substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, expression and/or activity of the gene product. In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, or a functional equivalent, may be inserted into appropriate expression vector to produce a transgene, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

Vectors can be BAC, plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the herpes thymidine kinase promoter, the regulatory sequences of the metallothionein gene (Brinster et al, 1982, Nature 296:3942), etc. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).

Generally, delivery of polynucleotides encoding transgenes for both ex vivo and in vitro applications can be accomplished by, for example, d extra n -mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, direct microinjection of the DNA into nuclei, and viral-mediated, such as adenovirus (and adeno-associated virus) or alphavirus, all well known in the art. Illustrative, but non-limiting methods of polynucleotide and polypeptide delivery are further discussed below. In certain embodiments, it will be preferred to deliver one or more transgenes to a cell using a viral vector or other in vivo or ex vivo polynucleotide delivery technique. In one preferred illustrative embodiment, the viral vector is a non-integrating vector. This may be achieved using any of a variety or well-known approaches, several of which are outlined below for purposes of illustration.

1 . Adenovirus Vectors

One illustrative method for in vivo delivery of one or more polynucleotide sequences involves the use of an adenovirus expression vector. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein in a sense or antisense orientation. Of course, in the context of an antisense construct, expression does not require that the gene product be synthesized.

Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al., 1991 ; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet & Perricaudet, 1991 ; Stratford-Perricaudet et ai, 1990; Rich et al., 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991 ; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz & Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al., 1993).

2. Retrovirus Vectors

The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants.

Recent advances have provided episomal forms of retroviral vectors based on lentiviruses {e.g., a type of retrovirus) that have become potent tools for efficient gene transfer to multiple cell types both in vitro and in vivo. In part this is attributable to the stability of transduction afforded by integration into the target cell genome. Nonintegrating vectors retain the high transduction efficiency and broad tropism of conventional lentiviruses but avoid the potential problems associated with the nonspecific integration of a transgene. In this respect they are particularly useful from a safety standpoint, and in certain embodiments, are preferred.

3. Adeno-Associated Virus Vectors

AAV (Ridgeway, 1988; Hermonat & Muzycska, 1984) is a parovirus, discovered as a contamination of adenoviral stocks. It is a ubiquitous virus (antibodies are present in 85% of the US human population) that has not been linked to any disease. It is also classified as a dependovirus, because its replication is dependent on the presence of a helper virus, such as adenovirus. Five serotypes have been isolated, of which AAV-2 is the best characterized. AAV has a single-stranded linear DNA that is encapsidated into capsid proteins VP1 , VP2 and VP3 to form an icosahedral virion of 20 to 24 nm in diameter (Muzyczka & McLaughlin, 1988).

AAV is also a good choice of delivery vehicles due to its safety, i.e., genetically engineered (recombinant) does not integrate into the host genome. There is a relatively complicated rescue mechanism: not only wild type adenovirus but also AAV genes are required to mobilize rAAV. Likewise, AAV is not pathogenic and not associated with any disease. The removal of viral coding sequences minimizes immune reactions to viral gene expression, and therefore, rAAV does not evoke an inflammatory response. 4. Other Viral Vectors as Expression Constructs

Other viral vectors may be employed as expression constructs in the present invention for the delivery of oligonucleotide or polynucleotide sequences to a host cell. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Coupar et al., 1988), polioviruses and herpes viruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Coupar et al., 1988; Horwich et al., 1990).

5. Non-Viral Methods

In order to effect expression of the oligonucleotide or polynucleotide sequences of the present invention, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming or transfecting cells lines, or in vivo or ex vivo , as in the treatment of certain disease states. As described above, one preferred mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle. In another embodiment, cells of the invention may be microinjected with polynucleotides of the present invention. In one embodiment, DNA microinjection is performed using borosilicate glass microinjection capillaries. In another preferred embodiment, DNA microinjection is accomplished using carbon nanotubes. In related embodiments, the polynucleotides of the invention are transferred to cells via electroporation. In other related embodiments, liposomes act as gene delivery vehicles and are described in U.S. Patent No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. 14:241 1 (1994), and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91 :1 1581 -1 1585.

Further embodiments provide additional non-viral delivery suitable for use in the methods of the present invention, including but not limited to mechanical delivery systems such as the approach described in Woffendin et ai, Proc. Natl. Acad. Sci. USA 91 (24):1 1581 (1994); deposition of

photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Patent No. 5,206,152 and WO 92/1 1033); the use of a hand-held gene transfer particle gun (see, e.g., U.S. Patent No. 5,149,655); and the use of ionizing radiation for activating transferred gene (see, e.g., U.S. Patent No. 5,206,152 and WO 92/1 1033). G. Polypeptides and Peptidomimetics

As noted above, the present invention, in certain aspects, provides for the prevention, treatment, cure, amelioration, or mitigation of disease or injuries in humans by the administration of autologous, allogeneic or xenogeneic cells. In particular illustrative embodiments, the present invention provides therapeutic compositions and methods of using the same to effect cell, tissue, and or organ regenerative, preventative, restorative, and/or ameliorative therapy. After therapy has been provided the cells can then be eliminated from the individual, as described elsewhere herein.

As used herein, the terms "polypeptide" and "protein" are used interchangeably, unless specified to the contrary, and according to conventional meaning, i.e., as a sequence of amino acids.

Polypeptides are not limited to a specific length, e.g., they may comprise a full length protein sequence or a fragment of a full length protein, and may include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring. Polypeptides of the invention may be prepared using any of a variety of well known recombinant and/or synthetic techniques, illustrative examples of which are further discussed below.

As used herein, "amino acid residue" refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are generally in the "L" isomeric form. Residues in the "D" isomeric form can be substituted for any L- amino acid residue, as long as the desired functional property is retained by the polypeptide. NH2 refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide. In keeping with standard polypeptide nomenclature described in J. Biol. Chem., 243:3552-59 (1969) and adopted at 37CF.R. §§ 1 .821 -1 .822, abbreviations for amino acid residues are shown in Table 1 : TABLE 1 - TABLE OF AMINO ACID NOMENCLATURE

A polypeptide variant may differ from a naturally occurring polypeptide in one or more substitutions, deletions, additions and/or insertions. Such variants may be naturally occurring or may be synthetically generated, for example, by modifying one or more of the above polypeptide sequences used in the methods of the invention and evaluating their effects using any of a number of techniques well known in the art.

In certain embodiments, a variant will contain conservative substitutions. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.

Modifications may be made in the structure of the polynucleotides and polypeptides of the present invention and still obtain a functional molecule that encodes a variant or derivative polypeptide with desirable characteristics. When it is desired to alter the amino acid sequence of a polypeptide to create an equivalent, or even an improved, variant polypeptide of the invention, one skilled in the art, for example, can change one or more of the codons of the encoding DNA sequence, e.g., according to Table 2.

TABLE 2- AMINO ACID CODONS

Amino Acids Codons

Methionine AUG

Asparagine AAC AAU

Proline CCA CCC CCG ecu

Glutamine CAA CAG

Arginine AGA AGG CGA CGC CGG CGU

Serine AGC AGU UCA UCC UCG UCU

Threonine ACA ACC ACG ACU

Valine GUA GUC GUG GUU

Tryptophan UGG

Tyrosine UAC UAU

Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity can be found using computer programs well known in the art, such as DNASTAR™ software. Preferably, amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cystine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

In a peptide or protein, suitable conservative substitutions of amino acids are known to those of skill in this art and generally can be made without altering a biological activity of a resulting molecule. Those of skill in this art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The

Benjamin/Cummings Pub. Co., p.224).

In addition, pegylation of polypeptides and/or muteins is expected to provide improved properties, such as increased half-life, solubility, and protease resistance. Pegylation is well known in the art.

Polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support. For example, a polypeptide may be conjugated to an

immunoglobulin Fc region.

In certain embodiments of the invention, there are provided fusion polypeptides, and polynucleotides encoding fusion polypeptides. Fusion polypeptide and fusion proteins refer to a polypeptide of the invention that has been covalently linked, either directly or via an amino acid linker, to one or more heterologous polypeptide sequences (fusion partners). The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N- terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order.

Fusion polypeptides of the present invention include any number and combination of polypeptides, polypeptide spacer moieties, and tethering moiety polypeptides, as described elsewhere herein.

The fusion partner may be designed and included for essentially any desired purpose provided they do not adversely affect the desired activity of the polypeptide. For example, in one embodiment, a fusion protein may be designed to encode multiple polypeptides as described herein, from a single transcript. In another embodiment, a fusion partner comprises a sequence that assists in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Other fusion partners may be selected so as to increase the solubility of the protein or to enable the protein to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the protein.

Fusion polypeptides may be produced by chemical synthetic methods or by chemical linkage between the two moieties or may generally be prepared using other standard techniques. In particular embodiments, it is preferred that fusion polypeptides are produced by fusion of a coding sequence of a polypeptide, a polypeptide spacer, and a coding sequence of tethering moiety polypeptide, as described elsewhere herein.

In certain embodiments, a fusion polypeptide comprises an antibody or antigen binding fragment thereof and at least the transmembrane domain or membrane spanning portion thereof or a cell surface receptor.

In further embodiments, a fusion polypeptide comprises an Fc domain and at least the transmembrane domain or membrane spanning portion thereof or a cell surface receptor.

A peptide linker (i.e., polypeptide spacer moiety) may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures, if desired. Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art. Certain peptide linker sequences may be chosen based on the following factors:

(1 ) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751 ,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference. The two coding sequences can be fused directly without any linker or by using a flexible polylinker composed of the pentamer Gly-Gly-Gly- Gly-Ser repeated 1 to 3 times. Such linker has been used in constructing single chain antibodies (scFv) by being inserted between VH and VL (Bird et ai, 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A.

85:5979-5883). The linker is designed to enable the correct interaction between two beta-sheets forming the variable region of the single chain antibody. Other linkers which may be used include Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu- Ser-Lys-Val-Asp (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. U.S.A.

87:1066-1070) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe- Arg-Ser-Leu-Asp (Bird et al., 1988, Science 242:423-426).

The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.

In general, polypeptides and fusion polypeptides (as well as their encoding polynucleotides) are isolated. An "isolated" polypeptide or

polynucleotide is one that is removed from its original environment. For example, a naturally-occurring protein is isolated if it is separated from some or all of the coexisting materials in the natural system. Preferably, such

polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.

H. Antibodies

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies {e.g., bispecific antibodies) formed from at least two intact

antibodies, and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment.

Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1 ) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS- PAGE under reducing or nonreducing conditions using, for example,

Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses

(isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three- dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

The terms "full length antibody," "intact antibody" and "whole antibody" are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

"Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CH1 ) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 1 13,

Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., PNAS USA 81 :6851 -6855 (1984)).

"Humanized" forms of non-human {e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human

immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et ai, Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma &

Immunol. 1 :105-1 15 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

"Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule {e.g., an antibody) and its binding partner {e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair {e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K_d). Affinity can be measured by common methods known in the art, including those described herein. Low- affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention.

Modifications in the biological properties of an antibody may be accomplished by selecting substitutions that affect (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids may be grouped according to similarities in the properties of their side chains (in A. L.

Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)):

It may be desirable to introduce one or more amino acid modifications in an Fc region of antibodies of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human lgG1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification [e.g., a substitution) at one or more amino acid positions including that of a hinge cysteine.

In accordance with this description and the teachings of the art, it is contemplated that in some embodiments, an antibody of the invention may comprise one or more alterations as compared to the wild type counterpart antibody, e.g., in the Fc region. These antibodies would nonetheless retain substantially the same characteristics required for therapeutic utility as compared to their wild type counterpart. For example, it is thought that certain alterations can be made in the Fc region that would result in altered (i.e., either improved or diminished) C1 q binding and/or Complement Dependent

Cytotoxicity (CDC), e.g., as described in WO99/51642. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No.

5,624,821 ; and WO94/29351 concerning other examples of Fc region variants. WO00/42072 (Presta) and WO 2004/056312 (Lowman) describe antibody variants with improved or diminished binding to FcRs. The content of these patent publications are specifically incorporated herein by reference. See, also, Shields et al. J. Biol. Chem. 9(2): 6591 -6604 (2001 ). Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et a/., J.

Immunol. 1 17:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1 q binding capability are described in U.S. Pat. No. 6,194,551 B1 , WO99/51642. The contents of those patent publications are specifically incorporated herein by reference. See, also, Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

In another aspect, the invention provides antibodies comprising modifications in the interface of Fc polypeptides comprising the Fc region, wherein the modifications facilitate and/or promote heterodimerization. These modifications comprise introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, wherein the protuberance is positionable in the cavity so as to promote complexing of the first and second Fc polypeptides. Methods of generating antibodies with these modifications are known in the art, e.g., as described in U.S. Pat. No. 5,731 ,168.

Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Harlow and Lane,

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.

Monoclonal antibodies specific for an antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:51 1 -519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest).

A variety of routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody. I. Cell Culture Compositions

The compositions and methods of the present invention require, in some embodiments, the culture of cells, including cell-based compositions. As discussed herein throughout, the present compositions and methods are useful for ex vivo and in vivo cell-based therapies, which in some embodiments require cell culture in a pharmaceutically acceptable cell culture medium. A therapeutic culture, cell culture, culture system, or cell culture compositions comprising a cell-based composition of the present invention can be administered separately by enteral or parenteral administration methods or in combination with other suitable compounds to effect the desired treatment goals. In particular illustrative embodiments, a culture, cell culture, culture system, or cell culture composition of the present invention is administered at the same time, before, or after an implant comprising a biocompatible matrix.

In further illustrative embodiments, the biocompatible matrix is surgically implanted in a subject and then a therapeutic culture, cell culture, culture system, or cell culture composition comprising a cell-based composition of the present invention is directly administered to the location of the implant.

In related illustrative embodiments, a therapeutic culture, cell culture, culture system, or cell culture composition comprising a cell-based composition of the present invention is administered systemically, and the cell- based composition then migrates to the location of the implant.

J. Formulations and Pharmaceutical Compositions

The formulations and compositions of the invention may comprise cell-based composition compositions comprising of a combination of any number of cells, polypeptides, polynucleotides, transgenes, and small molecules, as described herein, formulated in pharmaceutically acceptable or physiologically-acceptable solutions {e.g., culture medium) for administration to a cell, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents as well, such as, e.g., other proteins or polypeptides or various pharmaceutically-active agents.

In a particular embodiment, a formulation or composition according to the present invention comprises a cell contacted with a

combination of any number of polypeptides, polynucleotides, and small molecules, as described herein. In a related embodiment, a formulation or composition according to the present invention comprises a cell-based composition comprising a combination of any number of components, as described herein, formulated in a pharmaceutically acceptable cell culture medium.

There is virtually no limit to other reagents that may also be included in the compositions, provided that the additional reagents do not adversely affect the desired regenerative therapy.

Exemplary formulations for ex vivo delivery may also include the use of various transfection agents known in the art, such as calcium phosphate, electoporation, heat shock and various liposome formulations (i.e., lipid- mediated transfection). Liposomes, as described in greater detail below, are lipid bilayers entrapping a fraction of aqueous fluid. DNA spontaneously associates to the external surface of cationic liposomes (by virtue of its charge) and these liposomes will interact with the cell membrane. By including a small amount of an anionic lipid in an otherwise cationic liposome the DNA can be incorporated into the internal surface of the liposome, thus protecting it from enzymatic degradation. In certain embodiments, liposome formulations may optimized for a particular target cell type, such as for pancreatic islet cells, CNS cells, PNS cells, cardiac muscle cells, skeletal muscle cells, smooth muscle cells, hematopoietic cells, bone cells, liver cells, an adipose cells, renal cells, lung cells, chondrocytes, skin cells, follicular cells, vascular cells, epithelial cells, immune cells, endothelial cells, and the like. To facilitate uptake into the cell as endosomes, certain embodiments may employ targeting proteins in liposomes, including, for example, anti-MHC antibody, transferrin, the Sendai virus or its F protein, in addition to other desirable targeting agents. It is appreciated that Sendai viral proteins allow the plasmid DNA to escape from the endosome into the cytoplasm, thus avoiding degradation. As an additional example, the inclusion of a DNA binding protein (e.g., 28 kDa high mobility group 1 protein) enhances transcription by bringing the plasmid into the nucleus. Certain embodiments may include incorporating the Epstein-Barr virus Ori p and EBNA1 genes in the plasmid to maintain the plasmid as an episomal element.

Certain formulations may employ the use of molecular conjugates, which consist of protein or synthetic ligands to which a DNA binding agent has been attached. Delivery to the cell can be improved by using similar techniques to those for liposomes. Exemplary targeting proteins include

asialoglycoprotein, the Vpr protein from HIV, transferrin, polymeric IgA, and adenoviral proteins.

In certain aspects, the present invention provides

pharmaceutically acceptable compositions which comprise a therapeutically- effective amount of one cell-based compositions, including, but not limited to stem and progenitor cells comprising one or more resistance, cell-suicide, and/or factor transgenes, as described herein, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents (e.g., pharmaceutically acceptable cell culture medium). Methods for the delivery of polynucleotide molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2:139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar; Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules. These protocols can be utilized for the delivery of virtually any polynucleotide molecule. Polynucleotide molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.

As described in detail below, the pharmaceutical compositions of the present invention that comprise a combination of one or more of: i) a cell; ii) a cytotoxic agent; and iii) a pharmaceutically acceptable cell culture medium; may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1 ) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled- release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid {e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1 ) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (1 1 ) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21 ) polyesters, polycarbonates and/or polyanhydrides; (22) a pharmaceutically acceptable cell culture medium; and (23) other non-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the compositions described herein may comprise one or more pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate,

lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al., (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1 -19)

The pharmaceutically acceptable salts of the subject modulating agents include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

The term "pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine,

ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil- soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the ingredient which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about 99 percent of active ingredient, about 1 percent to about 90 percent of active ingredient, about 10 percent to about 80 percent of active ingredient, about 25 percent to about 75 percent of active ingredient, about 30 percent to about 70 percent of active ingredient, about 40 percent to about 60 percent of active ingredient, or about 50 percent of active ingredient.

In another embodiment, the amount of active ingredient in a single dosage from that is required to produce a therapeutic effect is about .1 % active ingredient, about 1 % active ingredient, about 5 % active ingredient, about 10% active ingredient, about 15% active ingredient, about 20% active ingredient, about 25% active ingredient, about 30% active ingredient, about 35% active ingredient, about 40% active ingredient, about 45% active ingredient, about 50% active ingredient, about 55% active ingredient, about 60% active ingredient, about 65% active ingredient, about 70% active ingredient, about 75% active ingredient, about 80% active ingredient, about 85% active ingredient, about 90% active ingredient, or about 95% active ingredient or more.

In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention.

Methods of preparing these formulations or compositions include the step of bringing into association cells of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacian or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules, trouches and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example,

carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamer and sodium lauryl sulfate; (7) wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and (1 1 ) controlled release agents such as crospovidone or ethyl cellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above- described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols,

polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a modulating agent as provided herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active modulating agent may be mixed under sterile conditions with a

pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.

Sprays can additionally contain customary propellants, such as

chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing an agent in the proper medium. Absorption enhancers can also be used to increase the flux of the agent across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the agent in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more cell-based therapeutcs in combination with one or more pharmaceutically acceptable sterile isotonic aqueous (e.g., pharmaceutically acceptable culture medium) or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, pharmaceutically acceptable culture media, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.

Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or

intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly-

(orthoesters) and poly-(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.

When a cell-based composition and/ or an agent are administered as pharmaceutical preparations, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99%, 1 to 90%, 10 to 80%, 25 to 75%, 30 to 70%, 40 to 60%, or about 50% of active ingredient in combination with a pharmaceutically acceptable carrier.

The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.

The phrases "parenteral administration" and "administered parenterally" as used herein means -modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

The modulating agents herein may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the modulating agents of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are

formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the

pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular modulating agent of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a composition will be that amount which is the lowest dose effective to produce a therapeutic effect.

Administration of one or more compositions and/or agents, compounds, or drugs can be performed in a single composition or multiple compositions. The compositions can be administered at the same time at the same site, in a single composition, or "piggybacked". In another embodiment, the compositions can be administered at the same time at different sites, for example, a first composition comprising cells of the invention can be

administered parenterally and a second composition comprising another or the same composition can be administered orally. In related embodiments, one or more compositions and/or agents, compounds, or drugs are administered to a subject via a parenteral and/or oral route. In another embodiment, a composition comprising cells of the invention and compositions and/or agents, compounds, or drugs are are administered via a parenteral route.

An effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of the adjunct therapies used in combination with a cell- based composition in various embodiments of this invention, will range from about 0.000001 to about 1000 mg per kilogram, about 0.000005 to about 950 mg per kilogram, about 0.00001 to about 850 mg per kilogram, about 0.00005 to about 750 mg per kilogram, about 0.0001 to about 500 mg per kilogram, about 0.0005 to about 250 mg per kilogram, about 0.001 to about 100 mg per kilogram, about 0.001 to about 50 mg per kilogram, about 0.001 to about 25 mg per kilogram, about 0.001 to about 10 mg per kilogram, about 0.001 to about 1 mg per kilogram, about 0.005 to about 100 mg per kilogram, about 0.005 to about 50 mg per kilogram, about 0.005 to about 25 mg per kilogram, about 0.005 to about 10 mg per kilogram, about 0.005 to about 1 mg per kilogram, about 0.01 to about 100 mg per kilogram, about 0.01 to about 50 mg per kilogram, about 0.01 to about 25 mg per kilogram, about 0.01 to about 10 mg per kilogram, about 0.01 to about 1 mg per kilogram, about 0.05 to about 50 mg per kilogram, about 0.05 to about 25 mg per kilogram, about 0.05 to about 10 mg per kilogram, about 0.05 to about 1 mg per kilogram, about 0.1 to about 25 mg per kilogram, about 0.1 to about 10 mg per kilogram, about 0.1 to about 1 mg per kilogram, about 0.1 to about .5 mg per kilogram of body weight per day.

In another embodiment, adjunctive therapies are administered orally or parenterally to a subject at a dose of about 0.25 to 3 g per kg, about 0.5 to 2.5 g per kg, about 1 to 2 g per kg, about 1 .25 to 1 .75 g per kg or about 1 .5 g per kg of bodyweight per day.

In particular embodiments, adjunctive therapies are administered orally or parenterally to a subject at a dose of about 10 g per kg, about .25 g per kg, about .50 g per kg, about .75 g per kg, about 1 .0 g per kg, about 1 .25 g per kg, about 1 .50 g per kg, about 1 .75 g per kg, or about 2.00 g per kg of bodyweight per day.

In other related embodiments, adjunctive therapies are administered orally or parenterally to a subject at a dose of about 0.01 \ig to 1 mg per kg, about 0.1 to 100 [ig per kg, or about 1 to 10 g per kg or any increment of concentration in between. For example, in particular

embodiments, a cell-based composition is administered orally or parenterally to a subject at a dose of about 1 g per kg, about 2 g per kg, about 3 \ig per kg, about 4 ig per kg, about 5 [ig per kg, about 6 [ig per kg, about 7 g per kg, about 8 ig per kg, about 9 [ig per kg, or about 10 [ig per kg.

In particular embodiments, adjunctive therapies are administered orally or parenterally to a subject at a dose of about .005 \ig per kg, about .01 per kg, about 1 .0 [ig per kg, about 10 [ig per kg, about 50 [ig per kg, about 100 [ig per kg, about 250 [ig per kg, about 500 [ig per kg, or about 1000 [ig per kg

In certain embodiments, compositions of the present invention comprise an effective amount of a cell-based composition and optionally comprise one or more adjunctive therapies.

In certain embodiments of the present invention, compositions comprising a cell-based composition and optionally comprising one or more adjunctive therapies can further comprise sterile saline, Ringer's solution, Hanks Balanced Salt Solution (HBSS), or Isolyte S, pH 7.4, serum free cellular media, or another pharmaceutically acceptable medium {e.g., cell culture medium), as discussed elsewhere herein.

One of ordinary skill in the art would be able to use routine methods in order to determine the appropriate route of administration and the correct dosage of an effective amount of a cell-based composition for methods of the present invention. It would also be known to those having ordinary skill in the art to recognize that in certain therapies, multiple administrations of pharmaceutical compositions of the invention will be required to effect therapy. For example a composition may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5, years, 10 years, or more.

Moreover, multiple administrations of the same or different compositions of the present invention may be administered, multiples times, for extended periods of time, as noted above.

In another aspect, the present invention provides

pharmaceutically acceptable compositions which comprise a therapeutically- effective amount of one or more cell-based compositions as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1 ) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin, lungs, or mucous membranes; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or (8) nasally.

K. Implants and Matrices

In various illustrative embodiments, the invention provides implants comprising cell-based compositions that can be employed as removable cell-based therapies in mammals, for example, in the repair, regeneration, or replacement of a cell, tissue, or organ.

As used herein, the term "implant" refers to a biocompatible natural and/or synthetic structure comprising one or more cell-based

compositions, cells, tissues, polymers, polynucleotides, magnetic particles, agarose particles, plastic particles, polypeptides, oligosaccharides, lipids, small molecules, lattices, and/or matrices that is injected or engrafted within a patient or subject that is suitable for directing or attracting a cell-based composition to repair, regenerate, or replace a cell, tissue or organ in vivo. In various embodiments, an implant refers to a matrix, as defined herein, that is suitable for directing or attracting a cell-based composition to repair, regenerate, or replace a cell, tissue or organ in vivo in a patient. At any point after therapy has occurred, the genetically modified cells of a cell-based composition may be removed or eliminated from the patient, leaving behind the implant and remaining cells.

As used herein, the term "matrix" refers to a biocompatible natural and/or synthetic environment that is suitable for directing or attracting a cell- based composition to repair, regenerate, or replace a cell, tissue or organ in vivo. Components of a natural or synthetic matrix, include but are not limited to, any number or combination of cells, tissues, polymers, polynucleotides, magnetic particles, agarose particles, plastic particles, polypeptides,

oligosaccharides, lipids, or small molecules.

It would be appreciated by one having skill in the art that an implant can comprise any of the matrices described herein, with any additional components or added features as described herein.

In particular illustrative embodiments, an implant comprises a biocompatible matrix that can be molded into any suitable form and has especially important roles to prepare tissues in a three-dimensional shape having a certain depth or height. Biomaterial science is an established and evolving field (Takayama et al, Principles of Tissue Engineering, Second

Edition, edit Lanza RP, Langer R, Vacanti J., Academic Press, San Diego, 2000, pg 209-218; Saltmann et al, Principles of Tissue Engineering, Second Edition, edit Lanza RP, Langer R, Vacanti J., Academic Press, San Diego, 2000, p 221 -236; Hubbell et al, Principles of Tissue Engineering, Second Edition, edit Lanza RP, Langer R, Vacanti J., Academic Press, San Diego, 2000, p 237-250; Thomson et al, Principles of Tissue Engineering, Second Edition, edit Lanza RP, Langer R, Vacanti J., Academic Press, San Diego, 2000, p 251 -262; Pachence et al, Principles of Tissue Engineering, Second Edition, edit Lanza RP, Langer R, Vacanti J., Academic Press, San Diego, 2000, p 263-278).

Chemists have developed methods to synthesize implants comprising biocompatible matrices comprising polymers to direct and modulate cell growth in vitro, ex vivo, and in vivo. The physical properties of the polymers can be modulated to create solid and liquid matrices of specific strengths and viscosities. Some polymers are stable in vivo and will remain in a patient's body for up to 1 , 2, 3, 4, 5, 10, 15 or more years. Other polymers are also biodegradable, resorbing at a fixed rate over time to allow replacement by newly synthesized extracellular matrix proteins. Resorption can occur within days to weeks or months following implantation (Pachence et al, Principles of Tissue Engineering, Second Edition, edit Lanza RP, Langer R, Vacanti J., Academic Press, San Diego, 2000, p 263-278).

In other illustrative embodiments, an implant comprises a biocompatible matrix comprising a bioabsorbable material. A porous carrier is preferably made of one component or a combination of multiple components selected from the group consisting of collagen, collagen derivatives, hyaluronic acid, hyaluronates, chitosan, chitosan derivatives, polyrotaxane, polyrotaxane derivatives, chitin, chitin derivatives, gelatin, fibronectin, heparin, laminin, and calcium alginate; wherein a support member is made of one component or a combination of multiple components selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone, polylactic acid-polyglycolic acid copolymer, polylactic acid-polycaprolactone copolymer, and polyglycolic acid-polycaprolactone copolymer (see, for example, U.S. Patent Nos.

5,077,049 and 5,42,033, and U.S. Patent Application Publication No.

2006/0121085, of which the polymer formulations and methods of making the same of each patent and application is incorporated herein in its entirety).

In further illustrative embodiments, an implant comprises a biocompatible matrix that includes: metals such as titanium, titanium alloys, stainless steels, cobalt-chromium alloys, and cobalt-chromium-molybdenum alloys; ceramics such as alumina ceramics, carbon ceramics, zirconia ceramics, silicon carbide ceramics, silicon nitride ceramics; and glass ceramics, and other bioinert materials also applicable to the support material of the matrix or lattice. Bioactive matrix materials like hydroxyapatite, calcium phosphate, calcium carbonate, and bioglass are further applicable to the support material of the matrix.

In particular illustrative embodiments of the present invention, the biocompatible matrix comprises a viscous, biocompatible liquid material. The biocompatible liquid is capable of gelling at body temperature and is selected from the group consisting of alginate, collagen, fibrin, hyaline, or plasma. The viscous, biocompatible liquid material can also be combined with a malleable, three dimensional matrix capable of filling an irregular tissue defect. The matrix is a material including, but not limited to, polyglycolic-polylactic acid, poly- glycolic acid, poly-lactic acid, or suture-like material.

The present invention also contemplates, in part, particular illustrative embodiments comprising an implant that further comprises a matrix comprising at least one population of non-genetically modified cells.

In certain illustrative embodiments, a non-genetically modified cell or population of non-genetically modified cells is cultured in or seeded onto a biocompatible matrix {e.g., material), or implant, such as one that includes extracellular matrix material, synthetic polymers, cytokines, growth factors, etc.

Alternatively, or in addition, particular illustrative embodiments of the present invention provide non-genetically modified cells that natively express the desired extracellular material, cytokine, and/or growth factor to promote or facilitate the repair, regeneration, or replacement of a cell, tissue, or organ.

In further illustrative embodiments, implants comprising matrices can be molded into desired shapes {e.g., three-dimensional structures) conducive to or facilitating cell, tissue, and/or organ development. The implant can be formed from polymeric material, having fibers such as a mesh or sponge. Such a structure provides sufficient area on which the cells can grow and proliferate. Desirably, the implant and matrix are biodegradable over time, so that they will be absorbed into the animal matter as it develops. Suitable polymers can be homopolymers or heteropolymers and can be formed from monomers including, but not limited to glycolic acid, lactic acid, propyl fumarate, caprolactone, and the like. Other suitable polymeric material can include a protein, polysaccharide, polyhydroxy acid, polyorthoester, polyanhydride, polyphosphozene, or a synthetic polymer, particularly a biodegradable polymer, or any combination thereof.

In another embodiment, an implant comprises a biocompatible matrix that comprises hormones, such as growth factors, cytokines,

morphogens {e.g., retinoic acid etc), extracellular matrix materials {e.g., fibronectin, laminin, collagen, etc.) or other materials {e.g., DNA, viruses, other cell types, etc.) to facilitate the proliferation and/or differentiation of the cell- based composition along a particular developmental pathway to repair, regenerate, or replace a cell, tissue, or organ.

In related illustrative embodiments, an implant comprises a biocompatible matrix comprising a hydrogel formed by cross-linking a polymer suspension that includes one or more homing cells dispersed therein. Without wishing to be bound to any particular theory, the foregoing method of implant formation permits the homing cells to be evenly dispersed throughout the matrix, facilitating more even permeation of the matrix with the cells. Of course, the homing cells within the matrix can natively express or be genetically modified to express factors that promote the repair, regeneration, or

replacement of a cell, tissue, or organ.

One having ordinary skill in the art will appreciate that implants can comprise matrices derived from any suitable source, e.g., Matrigel™ and can of course include commercial sources of suitable matrices. Another suitable matrix can be derived from the acellular portion of adipose tissue, muscle tissue, nervous system tissue, bone marrow tissue, and the like {i.e., other human tissue acellular matrices). Typically such matrices include one or more of proteins such as proteoglycans, glycoproteins, hyaluronin, fibronectins, collagens, and the like; all of which serve as excellent substrates for cell growth. Additionally, matrices can include hormones, cytokine, growth factors, and the like.

In one embodiment, the implant comprises a matrix characterized by an elongated lumen or networks of lumens that are functionally equivalent to stents, catheters, tubes, and the like. In another embodiment, the implant comprises a matrix characterized by a network of lumens, ducts, and/or pores that are functionally equivalent to naturally occurring vasculature of the tissue formed by the implanted cells and which is further lined with endothelial cells.

In another embodiment, the implant comprises a synthetic blood vessel. Illustrative synthetic blood vessels implants and methods of preparing the same are discussed in U.S. Patent Application Publication Nos.

2004/0068065 and 2008/0233164, each of which is included herein by reference in its entirety. In another embodiment, the matrix is further coupled to blood vessels or other ducts at the time of implantation to form a vascular or ductile network throughout the matrix. Free-form fabrication techniques refer to any technique known in the art that builds a complex 3-dimensional object as a series of 2-dimensional layers. Such methods can be adapted for use with a variety of polymeric, inorganic and composite materials to create structures with defined compositions, strengths and densities. Thus, utilizing such methods, precise channels and pores can be created within the matrix to control subsequent cell growth and proliferation within the matrix of one or more cells types having a defined function. In such a way, homing cells corresponding to the various types of a particular organ's cells can be combined to form a partial structure or an entire structure. Such cells are combined in the matrix to provide a vascular network lined with endothelial cells interspersed throughout the cells.

In another embodiment, the implant can be molded into virtually any structure including, but not limited to, a solid implant, a semi-solid implant, a liquid implant, a porous implant, a stent, a catheter, and a synthetic vein. In various illustrative embodiments, an implant comprising the inventive cell-based compositions and biocompatible matrices support tissue engineering and regeneration in a patient or subject. Thus, the present invention contemplates, in part, to use an implant comprising a cell-based composition in combination with a matrix, incorporating any of the features disclosed herein. The exact nature of the implant will vary according to the use desired. The implant can comprise mature tissue or can include immature tissue or the lattice or matrix. Such an implant is injected or engrafted within a patient or subject to encourage the generation or regeneration of mature tissue within the patient or subject.

In various illustrative embodiments, methods of directing a cell- based composition to a site in vivo comprise implanting a biocompatible scaffold, such as a biocompatible matrix, into the individual at or near the site of cell, tissue, or organ injury, wherein the desired regenerative, restorative, preventative, or ameliorative therapy is desired and/or required.

For example, an implant comprising a biocompatible matrix can be implanted in pancreatic tissue, neural tissue, cardiac tissue, bone marrow, muscle tissue, bone tissue, skin tissue, liver tissue, hair follicles, vascular tissue, adipose tissue, lung tissue, retinal tissue, corneal tissue, and kidney tissue, as required to direct the appropriate therapy.

In particular illustrative embodiments, an implant comprises one or more components selected from the group consisting of: a genetically modified cell, a non-genetically modified cell, a polymer, a biologically inert particle, a polypeptide, an oligosaccharide, a lipid, a polynucleotide, a small molecule, a lattice, and a biocompatible matrix or any combination thereof.

In particular illustrative embodiments, the biocompatible matrix comprises one or more components selected from the group consisting of: a homing cell, polymer, a biologically inert particle, a polypeptide, an

oligosaccharide, a lipid, a polynucleotide, and a small molecule or any combination thereof. L. Methods of Use

In various illustrative embodiments of the invention, a cell-based composition is administered to an individual, wherein the composition

comprises one or more cytotoxic agents and at least one plurality of genetically modified cells, wherein the genetically modified cells are resistant to the one or more cytotoxic agents; are directed to a site in vivo; and allowed to remain at the site under conditions and for a time sufficient to provide an amount of therapy to the individual; and once therapy has been achieved, apoptosis or cell suicide is induced in the genetically modified cells to eliminate the cells from the individual. The apoptosis or cell suicide program is specific to the genetically modified cells and does not target non-target cells to undergo programmed cell death.

According to several illustrative embodiments of the present invention, cell-based compositions comprising stem and/or progenitor cells have the ability to give rise to many specialized cells in an organism. Without wishing to be bound to any particular theory, the advantages of certain illustrative embodiments of the present invention are the localization of the cell- based composition to the site of therapy in vivo; the ability for the cells to provide therapy; and the removal of the cells from the individual being treated.

Cell-based compositions can be used to treat many cardiovascular diseases for which therapy is currently inadequate. For example, cell-based compositions directed or localized to a given heart type of heart cells could potentially be used to repair the failing heart when it can no longer pump, to generate growth of heart chambers when infants are born with malformed hearts, and to repair vascular damage resulting from high blood pressure and atherosclerosis. Thus, in one embodiment, cell-based

compositions of the present invention are transplanted into the heart,

successfully repopulate the heart tissue and work together with the host cells to repair or replace damaged tissue. After the therapy has been provided, the cell-based compositions can be eliminated from the individual. One having ordinary skill in the art would appreciate that upon localization of a cell-based composition comprising a stem cell, a progenitor cell, or a reprogrammed cell to a site in vivo, the cell based composition can be induce to produce one or more growth factors, cytokines, and/or differentiation factors that induce local populations of adult stem cells to differentiate into the desired cell type. Differentiation can occur by contacting the cells with one or more differentiation agents, or by virtue of locating a stem cell, a progenitor cell, or a reprogrammed cell to the site of therapy, wherein differentiation can proceed naturally.

Cell-based compositions of the present invention can be engineered (e.g., programmed) to recognize specialized cell types such as bone, cartilage and salivary cells; thus, directing or localizing the cell-based composition to damaged cells, tissues, or organs for use as a regenerative therapy. Examples include the treatment of temporomandibular joint disorders (TMDs), the replacement of skeletal elements lacking or damaged in diseases such as fibrous dysplasian of bone using cells grown in special natural or synthetic scaffolding materials, and the replacement of salivary cells damaged by disease (Sjogren's Syndrome) or radiation for head and neck cancer.

Cell-based compositions comprise cells that can be differentiated into highly important tissue-specific cells. For example, cell-based

compositions comprising the appropriate type and potency of stem and/or progenitor cells can be differentiated into pancreatic islet beta cells, which is are capable of secreting insulin. Isolated cells of this type are used for

transplantation studies and, to a limited extent, in human therapeutic

approaches to treat type 1 diabetes.

Other examples include cellular therapy to replace diseased liver tissue. In this case, a cell-based composition that comprises cells of the appropriate potency is directed to the liver and is subsequently differentiated or programmed along the cell lineage of a functional liver cell. Other examples could include various forms of kidney cells or potentially bladder cells. Various embodiments of the present invention contemplate, in part, that there are numerous other examples in addition to diabetes, liver failure, kidney failure, and urologic diseases in which cell-based compositions of the present invention will have a major therapeutic role.

A cell-based composition that comprises cells of the appropriate potency is suitable to treat and/or ameliorate the many diseases that result from the loss of nerve cells, and mature nerve cells that cannot normally divide to replace those that are lost. In Parkinson's disease, nerve cells of the substantia nigra that make the chemical dopamine die. In Alzheimer's disease, cells that make acetylcholine die. In amyotrophic lateral sclerosis the motor nerve cells that activate muscles die. In stroke, brain trauma, and spinal cord injury many types of cells are lost. There are many more disorders that affect both adults and young children in which nerve cells die.

In some diseases, a cell-based composition that comprises cells of the appropriate potency can provide therapy for a particular type of nerve cell— a different kind of nerve cell for Parkinson's than for Alzheimer's than for amyotrophic lateral sclerosis and so on. For other disorders, like multiple sclerosis, it is not nerve cells, but supporting cells, the glial cells that wrap electrical insulation around nerve fibers, that a cell-based composition will replace. In other neurological insults, for example brain trauman or stroke, cell- based compositions comprise cells that can be used to regenerate regions of brain tissue, with many integrated types of brain cells.

In various illustrative embodiments, the present invention provides methods of administering a cell-based composition near an injured or diseased cell, tissue, and/or organ in need of regenerative therapy. Thus, methods administering the foregoing cell-based compositions could lead to cures for diseases that require treatment through transplantation, including autoimmune diseases. (Autoimmune diseases include multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, and type-l diabetes).

For disorders affecting the nervous system, such as Alzheimer's and Parkinson's diseases, amyotrophic lateral sclerosis, and spinal cord and brain injury, directing a cell-based composition to the injury site provides for replacing cells lost in these conditions and of recovery of function.

A cell-based composition comprising cells of the appropriate potency (e.g., stem and/or progenitor cells) can be localized to any particular diseased type of bone or cartilage cell or tissue; thus, a cell-based composition of the present invention can lead to the long-term correction of many diseases and degenerative conditions in which bone or cartilage cells are deficient in numbers or defective in function. Such an approach is an important therapeutic option for genetic disorders of bone and cartilage, such as osteogenesis imperfecta and the various chondrodysplasias.

Particular illustrative embodiments of the present invention further contemplate, in part, that a cell-based composition directed to the inner ear can be used to replace the sound-detecting hair cells in the inner ear that are often lost due to genetic, infectious, traumatic, or pharmacologic causes.

There is good evidence that many of the mental and behavioral disorders such as schizophrenia, autism, manic-depressive illness and memory disorders, result from permanent disruption of brain circuitry or brain chemistry. Thus, in one embodiment, a cell-based composition localized to the disrupted cells responsible for such disorders can be used to correct such defects and restore mental health to the subject. Similar transplant strategies apply to other severe developmental disorders, such as autism.

A cell-based composition can also provide regenerative therapy to particular regions of the nervous system and also provide a means of replacing neurons destroyed by drug abuse. This is especially useful for individuals who have abused drugs such as methamphetamine, MDMA (ecstacy) and inhalants which have been shown in animal and some human studies to cause long-term, possibly permanent damage to selected areas of the brain. For example, recent research has shown that methamphetamine can have significant toxic effects on dopaminergic and serotonergic neurons in the brain. This is of particular concern because of the spreading use of this drug and may be related to the dramatic behavioral effects, including the development of psychotic-like behavior patterns that methamphetamine can have in some people.

Alcohol is a major source of damage to organs, such as the liver and brain, that may or may not regain function with abstinence from drinking. Development of medications that accelerate recovery in organs damaged by alcohol would be a major breakthrough. Such an advance would lessen human suffering and the economic burden associated with alcohol-induced organ damage. For cases of irreversible organ damage a cell-based composition directed to the affected tissue can be used to facilitate generation of new organ tissue.

As noted herein, the present invention relates, in part, to methods and compositions to be used as cell-based therapies in a wide variety of disease, disorders, or conditions in which the replacement, regeneration, expansion, and/or maintenance of a cell, tissue, and/or organ; and subsequent elimination after therapy has been provided is desirable or beneficial in treating or reducing the symptoms associated with the disease, disorder or condition.

The methods provided herein are contemplated for use with in vivo and ex vivo therapeutic modalities, alone or in combination with each other. For example, in certain embodiments, in vivo therapeutic modalities may involve localized, in vivo administration, such as direct injection of one or more cell-based compositions (including cell culture based compositions, as described elsewhere herein), into a subject, or into a biocompatible material {e.g., an implant) or into a target tissue or target organ of a subject. Optionally, the composition comprises one or more growth factors, cytokines or adjunctive therapies that can be administered with the cell-based composition.

In other embodiments, in vivo therapeutic modalities may comprise system administration of one or more cell-based compositions (including cell culture based compositions, as described elsewhere herein). Particular modes of in vivo administration are exemplified elsewhere herein and known to a person skilled in the art. In certain aspects relating to ex vivo therapy, cells from one or more tissues may be isolated, for example, from the subject to be treated, from another subject, from a tissue culture source, or from any other desirable source of cells.

Illustrative cells, tissues, or organs, to be repaired and or regenerated (i.e., targeted) include, but are not limited to, neural cells in tissues (e.g., to treat ischemic injury, spinal cord injury), cardiac cells or tissues (e.g. , to treat myocardial infarction or other ischemic injury, congestive heart failure), pancreatic islet cells or pancreatic tissues (e.g., to treat diabetes, such as Type II diabetes), motor neuron cells (e.g. , to treat to Parkinson's Disease and provide motor neuron cell regeneration), hepatocyte cells or tissues, renal cells (e.g., to treat liver or kidney transplant and provide liver or kidney regeneration), lung cells or tissues, skin tissues (e.g., to improve wound healing, and provide skin transplants for burn therapy), skeletal muscle tissue, hematopoietic cell transplant, expansion, and/or regeneration (e.g. , B-cell regeneration and replacement, immature progenitor cell expansion, reprogramming red blood cell fate to white blood cell fate, modulate homing and engraftment), hair follicles (e.g., improve hair growth), among others known to a person skilled in the art.

Methods of the present invention are suitable for providing therapy to the hematopoietic cell system including, but not limited to, altering the types of hematopoietic cells generated following a transplant by

programming a cell toward a desired lineage, such as red blood cells, platelets, B-cells, T-cells, or other specialized immune or hematopoietic cells. For example, individuals with myelodysplastic syndrome suffer from ineffective production of red blood cells, such that altering hematopoietic stem/progenitor cell potency by programming a hematopoietic stem/progenitor cells to red blood cells would provide a beneficial treatment for this condition.

In certain embodiments, administration of a cell-based composition or implant comprising the same can be performed surgically as part of a tissue or organ transplant, such as a liver transplant, heart transplant, neural tissue transplant, kidney transplant, bone marrow transplant, stem cell transplant, skin transplant, or lung transplant.

In certain illustrative embodiments, cell-based compositions and methods of using the same provided herein may be employed to treat neurodegenerative or neurological conditions or disease, including, for example, Alzheimer's disease, amyotrophic lateral sclerosis, ataxia

telangiectasia, HIV associated dementia, Huntington's disease, multiple sclerosis, multiple system atrophy, Parkinson's disease, paralysis, Pick's disease, schizophrenia, spinal muscular atrophy, stroke, and prion disease.

In certain embodiments, cell-based compositions and methods of using the same provided herein may be utilized to treat or manage the symptoms of degenerative muscle diseases, such as muscular dystrophy, duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, myotome muscular dystrophy, congenital myopathy, or mitochondrial myopathy.

In certain embodiments, the methods provided herein may be utilized to treat or manage the symptoms of degenerative cardiovascular diseases or conditions, such as aneurysms, angina, arryhthmias,

atherosclerosis, cardiomyopathy, cerebrovascular disease, congenital heart disease, congestive heart failure, myocarditis, valve disease, dilated

cardiomyopathy, myocardial infarction (heart attack), hypertrophic

cardiomyopathy, restrictive cardiomyopathy, venous thromboembolism, vascular restenosis, or coronary artery disease with resultant ischemic cardiomyopathy.

In other embodiments, the methods provided herein may be utilized to treat or manage the symptoms of degenerative liver diseases, such as nephritic disease, cirrhosis, alcoholic cirrhosis, fatty liver, alcoholic hepatitis, viral hepatitis, liver carcinoma, post necrotic cirrhosis, biliary cirrhosis, hepatocellular injury or a biliary tract disorder.

Certain embodiments encompass the treatment of degenerative pancreatic diseases, diabetes (e.g., Type I and Type II), diabetes related disorder, hyperglycennia, hyperinsulinaennia, hyperlipidaennia, insulin resistance, impaired glucose metabolism, obesity, diabetic retinopathy, macular

degeneration, cataracts, diabetic nephropathy, glomerulosclerosis, and diabetic neuropathy.

Certain embodiments encompass methods of increasing or improving cell or tissue regeneration in a subject, wherein the cell or tissue regeneration occurs in bone, chondrocytes/cartilage, muscle, skeletal muscle, cardiac muscle, pancreatic cells, endothelial cells, vascular endothelial cells, adipose cells, liver, skin, connective tissue, hematopoietic stem cells, neonatal cells, umbilical cord blood cells, fetal liver cells, adult cells, bone marrow cells, peripheral blood cells, erythroid cells, granulocyte cells, macrophage cells, granulocyte-macrophage cells, B cells, T cells, multipotent mixed lineage colony types, embryonic stem cells, mesenchymal stem/progenitor cells, mesodermal stem/progenitor cells, neural stem/progenitor cells, or nerve cells.

Other embodiments include methods of treating immune-related diseases, such as diabetes, graft vs. host disease, immunodeficiency disease, hematopoietic malignancy, hematopoietic failure, or hematopoietic stem cell transplantation.

Further embodiments include methods of treating degenerative diseases and other medical conditions that might benefit from regeneration therapies such atherosclerosis, coronary artery disease, obstructive vascular disease, myocardial infarction, dilated cardiomyopathy, heart failure, myocardial necrosis, valvular heart disease, mitral valve prolapse, mitral valve

regurgitation, mitral valve stenosis, aortic valve stenosis, and aortic valve regurgitation, carotid artery stenosis, femoral artery stenosis, stroke,

claudication, and aneurysm; cancer-related conditions, such as structural defects resulting from cancer or cancer treatments; the cancers such as, but not limited to, breast, ovarian, lung, colon, prostate, skin, brain, and

genitourinary cancers; skin disorders such as psoriasis; joint diseases such as degenerative joint disease, rheumatoid arthritis, arthritis, osteoarthritis, osteoporosis and ankylosing spondylitis; eye-related degeneration, such as cataracts, retinal and macular degenerations such as maturity onset; macular degeneration, retinitis pigmentosa, and Stargardt's disease; auralrelated degeneration, such as hearing loss; lung-related disorders, such as chronic obstructive pulmonary disease, cystic fibrosis, interstitial lung disease, emphysema; metabolic disorders, such as diabetes; genitourinary problems, such as renal failure and glomerulonephropathy; neurologic disorders, such as dementia, Alzheimer's disease, vascular dementia and stroke; and endocrine disorders, such as hypothyroidism.

Regeneration therapies from the cell-based compositions and methods of using the same are useful for traumas to skin, bone, joints, eyes, neck, spinal column, and brain, for example, that result in injuries that would normally result in scar formation.

In another illustrative embodiment, cell-based compositions and methods of using the same are useful for replacing epidermal cells, pancreatic parenchymal cells, pancreatic duct cells, hepatic cells, blood cells, cardiac muscle cells, skeletal muscle cells, osteoblasts, skeletal myoblasts, neurons, vascular endothelial cells, pigment cells, smooth muscle cells, fat cells, bone cells, and chondrocytes.

In a particular illustrative embodiment, cell-based compositions and methods of using the same are useful for replacing cells selected from a pancreatic islet cell, a CNS cell, a PNS cell, a cardiac cell, a skeletal muscle cell, a smooth muscle cell, a hematopoietic cell, a bone cell, a liver cell, an adipose cell, a renal cell, a lung cell, a chondrocyte, a skin cell, a follicular cell, a vascular cell, an eptithelial cell, an immune cell, and an endothelial cell.

In certain illustrative embodiments, cell-based compositions and methods of using the same are useful for replacing myocytes, chondrocytes, epithelial cells, or neurons.

In another illustrative embodiment, cell-based compositions and methods of using the same are useful for replacing tissue including, but not limited to, pancreatic tissue, neural tissue, cardiac tissue, bone marrow, muscle tissue, bone tissue, skin tissue, liver tissue, hair follicles, vascular tissue, adipose tissue, lung tissue, and kidney tissue. In a particular illustrative embodiment, methods and compositions of the invention are useful for replacing an organ is selected from the group consisting of brain, spinal cord, heart, liver, kidney, stomach, intestine, eye, and pancreas.

In another embodiment, cell-based compositions and methods of using the same are useful for ex vivo or in vivo treatment or prophylaxis of a disease or disorder due to a defect in a cell, tissue or organ of a subject.

In one illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the circulatory system (blood cells, etc.). Examples of the diseases, disorders, and conditions of the circulatory system include, but are not limited to, anemia {e.g., aplastic anemia (particularly, severe aplastic anemia), renal anemia, cancerous anemia, secondary anemia, refractory anemia, etc.), cancer or tumors {e.g., leukemia); and after

chemotherapy therefore, hematopoietic failure, thrombocytopenia, acute myelocytic leukemia (particularly, a first remission (high-risk group), a second remission and thereafter), acute lymphocytic leukemia (particularly, a first remission, a second remission and thereafter), chronic myelocytic leukemia (particularly, chronic period, transmigration period), malignant lymphoma (particularly, a first remission (high-risk group), a second remission and thereafter), multiple myeloma (particularly, an early period after the onset), and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the nervous system. Examples of such diseases, disorders, and conditions of the nervous system include, but are not limited to, dementia, cerebral stroke and sequela thereof, cerebral tumor, spinal injury, and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the immune system. Examples of such diseases, disorders, and conditions of the immune system include, but are not limited to, T-cell deficiency syndrome, leukemia, and the like. In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the motor organ or skeletal system. Examples of such diseases, disorders, and conditions of the motor organ and skeletal system include, but are not limited to, fracture, osteoporosis, luxation of joints, subluxation, sprain, ligament injury, osteoarthritis, osteosarcoma, Ewing's sarcoma, osteogenesis imperfecta, osteochondrodysplasia, and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the skin system. Examples of such diseases, disorders, and conditions of the skin system include, but are not limited to, atrichia, melanoma, cutis matignant lymphoma, hemangiosarcoma, histiocytosis, hydroa, pustulosis, dermatitis, eczema, and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the endocrine system. Examples of such diseases, disorders, and conditions of the endocrine system include, but are not limited to, hypothalamus/hypophysis diseases, thyroid gland diseases, accessory thyroid gland (parathyroid) diseases, adrenal cortex/medulla diseases, saccharometabolism abnormality, lipid metabolism abnormality, protein metabolism abnormality, nucleic acid metabolism abnormality, inborn error of metabolism (phenylketonuria, galactosemia, homocystinuria, maple syrup urine disease), analbuminemia, lack of ascorbic acid systhetic ability, hyperbilirubinemia, hyperbilirubinuria, kallikrein deficiency, mast cell deficiency, diabetes insipidus, vasopressin secretion abnormality, dwarfism, Wolman's disease (acid lipase deficiency)), mucopolysaccharidosis VI, and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the respiratory system. Examples of such diseases, disorders, and conditions of the respiratory system include, but are not limited to, pulmonary diseases (e.g., pneumonia, lung cancer, etc.), bronchial diseases, and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the digestive system. Examples of such diseases, disorders, and conditions include, but are not limited to, esophagial diseases (e.g., esophagial cancer, etc.), stomach/duodenum diseases {e.g., stomach cancer, duodenum cancer, etc.), small intestine diseases/large intestine diseases (e.g., polyps of the colon, colon cancer, rectal cancer, etc.), bile duct diseases, liver diseases (e.g., liver cirrhosis, hepatitis (A, B, C, D, E, etc.), fulminant hepatitis, chronic hepatitis, primary liver cancer, alcoholic liver disorders, drug induced liver disorders, etc.), pancreatic diseases (acute pancreatitis, chronic pancreatitis, pancreas cancer, cystic pancreas diseases, etc.), peritoneum/abdominal wall/diaphragm diseases (hernia, etc.), Hirschsprung's disease, and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the urinary system. Examples of such diseases, disorders, and conditions include, but are not limited to, kidney diseases (e.g., renal failure, primary glomerulus diseases, renovascular disorders, tubular function abnormality, interstitial kidney diseases, kidney disorders due to systemic diseases, kidney cancer, etc.), bladder diseases (e.g., cystitis, bladder cancer, etc.), and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the genital system. Examples of such diseases, disorders, and conditions include, but are not limited to, male genital organ diseases (e.g., male sterility, prostatomegaly, prostate cancer, testicular cancer, etc.), female genital organ diseases (e.g., female sterility, ovary function disorders, hysteromyoma, adenomyosis uteri, uterine cancer, endometriosis, ovarian cancer, villosity diseases, etc.), and the like.

In another illustrative embodiment, a cell-based composition of the invention is useful to treat disorders of the cardiac system. Examples of such diseases, disorders, and conditions include, but are not limited to, heart failure, angina pectoris, myocardial infarct, arrhythmia, valvulitis, cardiac muscle/pericardium diseases, congenital heart diseases (e.g., atrial septal defect, arterial canal patency, tetralogy of Fallot, etc.), artery diseases (e.g., arteriosclerosis, aneurysm), vein diseases (e.g., phlebeurysm, etc.),

lymphoduct diseases (e.g., lymphedema, etc.), and the like.

In various illustrative embodiments, a cell-based composition of the invention is suitable for treating/preventing cancer. For example, stem cells are important in the treatment of cancer based on the finding that cancer cells may have certain stem cell-like properties, specifically, the ability to renew themselves. Thus, the present invention contemplates, in part, to circumvent this property by directing a cell-based composition to the cancer cells in order to elicit cancer cell differentiation, along with subsequent surgical or

chemotherapeutic treatment to ensure removal of the treated cancer cells. In one illustrative embodiment, a cell-based composition used to treat cancer further comprises a transgene encoding a differentiation factor that causes cancer cells to differentiate.

Cancers that are suitable therapeutic targets of the present invention include cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, eye, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma;

carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined

hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp;

adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma;

basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma;

follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;

endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma;

medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma;

adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant;

myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant;

phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant;

dysgerminoma; embryonal carcinoma; teratoma, malignant; struman ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant;

ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant;

ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant;

neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non- Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia;

monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

As used herein, the term "cancer" (also used interchangeably with the terms, "hyperproliferative" and "neoplastic") refers to cells having the capacity for autonomous growth, i.e., an abnormal state or condition

characterized by rapidly proliferating cell growth. Cancerous disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, e.g., malignant tumor growth, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state, e.g., cell proliferation associated with wound repair. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. The term "cancer" includes malignancies of the various organ systems, such as those affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, non-small cell carcinoman of the lung, cancer of the small intestine and cancer of the esophagus. The term

"carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term

"carcinoma" also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An

"adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term

"sarcoma" is art recognized and refers to malignant tumors of mesenchymal derivation.

Examples of cellular proliferative and/or differentiative disorders of the lung include, but are not limited to, tumors such as bronchogenic

carcinoma, including paraneoplastic syndromes, bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchial carcinoid, miscellaneous tumors, metastatic tumors, and pleural tumors, including solitary fibrous tumors (pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders of the breast include, but are not limited to, proliferative breast disease including, e.g., epithelial hyperplasia, sclerosing adenosis, and small duct papillomas; tumors, e.g., stromal tumors such as fibroadenoma, phyllodes tumor, and sarcomas, and epithelial tumors such as large duct papilloma; carcinoman of the breast including in situ (noninvasive) carcinoma that includes ductal carcinoma in situ (including Paget's disease) and lobular carcinoma in situ, and invasive (infiltrating) carcinoma including, but not limited to, invasive ductal carcinoma, invasive lobular carcinoma, medullary carcinoma, colloid

(mucinous) carcinoma, tubular carcinoma, and invasive papillary carcinoma, and miscellaneous malignant neoplasms. Disorders in the male breast include, but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders involving the colon include, but are not limited to, tumors of the colon, such as non-neoplastic polyps, adenomas, familial syndromes, colorectal

carcinogenesis, colorectal carcinoma, and carcinoid tumors. Examples of cancers or neoplastic conditions, in addition to the ones described above, include, but are not limited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,

lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer, esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer, prostate cancer, uterine cancer, cancer of the head and neck, skin cancer, brain cancer, squamous cell carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,

choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular cancer, small cell lung carcinoma, non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,

hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposi sarcoma.

Contemplated useful secondary or adjunctive therapeutic agents in this context include, but are not limited to: chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine,

trietylenephosphoramide, triethiylenethiophosphoramide and

methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsachne; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;

pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;

methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; amninopterin; cyclosporine, sirolimus, rapamycin, rapalogs, ibandronate; topoisomerase inhibitor RFS 2000;

difluorometlhylornithine (DMFO); retinoids such as retinoic acid; CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU, leucovovin; anti- estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX® tamoxifen), raloxifene (EVISTA®), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY1 17018, onapristone, and toremifene (FARESTON®); anti-progesterones; estrogen receptor down- regulators (ERDs); estrogen receptor antagonists such as fulvestrant

(DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); aptamers, described for example in U.S. Patent No. 6,344,321 , which is herein incorporated by reference in its entirety; anti HGF monoclonal antibodies {e.g., AV299 from Aveo, AMG102, from Amgen); truncated mTOR variants {e.g., CGEN241 from Compugen); protein kinase inhibitors that block mTOR induced pathways {e.g., ARQ197 from Arqule, XL880 from Exelexis, SGX523 from SGX Pharmaceuticals, MP470 from Supergen, PF2341066 from Pfizer); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor {e.g., LURTOTECAN®); rmRH {e.g.,

Other compounds that are effective in treating cancer are known in the art and described herein that are suitable for use with the compositions and methods of the present invention are described, for example, in the

"Physicians Desk Reference, 62nd edition. Oradell, NJ: Medical Economics Co., 2008 ", Goodman & Gilman's "The Pharmacological Basis of Therapeutics, Eleventh Edition. McGraw-Hill, 2005", "Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.", and "The Merck Index, Fourteenth Edition. Whitehouse Station, NJ: Merck Research Laboratories, 2006", incorporated herein by reference in relevant parts

M. Kits

In various other embodiments, the present invention provides, in part, a kit for regenerative therapy. Kits of the invention comprise: one or more cytotoxic agents; a cell-based composition comprising a plurality of genetically modified cells, wherein the genetically modified cells comprise one or more resistance transgenes that provide resistance to the one or more cytotoxic agents and/or one or more inducible cell suicide transgenes and/or a

pharmaceutically acceptable cell culture medium.

In another embodiment, the present invention provides a kit comprising: an implant, wherein the implant comprises: a biocompatible matrix; one or more cytotoxic agents; a cell-based composition comprising a plurality of genetically modified cells, wherein the genetically modified cells comprise one or more resistance transgenes that provide resistance to the one or more cytotoxic agents and/or one or more inducible cell suicide transgenes and/or pharmaceutically acceptable cell culture medium.

In particular embodiments, the elements of the kit may be packaged separately, or together, or may comprise certain reagents packaged together and others packaged separately. For example, cells may be stored frozen in a separate container; while cytotoxic agents may be stored lyophilized at room temp; as a liquid at 4°C; or frozen.

In certain embodiments, the kit comprises components for administration, including, but not limited to needles, syringes, buffers, re- hydrating agents, diluents, anesthetics, and the like.

The present invention further contemplates that any components of a composition and/or implant described herein may be included, without limitation, in a kit of the invention. For example, cells, polynucleotides, polypeptides, implants, and pharmaceutical compositions can all be included within particular kits of the invention.

The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA

techniques, genetics, immunology, cell biology, stem cell protocols, cell culture and transgenic biology that are within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A

Laboratory Manual (3^rd Edition, 2001 ); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2^nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1 ^st Edition, 1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in

Molecular Biology, Greene Pub. Associates and Wiley-interscience; and Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be

understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of is meant to include the elements listed after the phrase, and is also limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific

embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

1 . A method of cell-based therapy comprising:

(a) administering to the individual:

(i) a plurality of genetically modified cells; and

(ii) one or more cytotoxic agents, wherein the cells are resistant to the cytotoxic agent,

(b) allowing the cells to remain at the site under conditions and for a time sufficient to provide an amount of therapy to the individual; and

(c) inducing apoptosis in the cells,

thereby providing cell-based therapy.

2. The method of claim 1 , wherein the cells are directed to a site in vivo.

3. The method of claim 1 , wherein the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell.

4. The method of claim 1 , wherein the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: a cell differentiated from an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell.

5. The method of claim 1 , wherein the plurality of genetically modified cells comprises at least one somatic cell.

6. The method of any one of claims 1 -5, wherein the plurality of genetically modified cells comprises a cell isolated from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, and kidney.

7. The method of any one of claims 1 -5, wherein the plurality of genetically modified cells comprises a mammalian cell.

8. The method of any one of claims 1 -5, wherein the plurality of genetically modified cells comprises a human cell.

9. The method of claim 1 , wherein each of the plurality of genetically modified cells comprises at least one resistance transgene that provides resistance to the one or more cytotoxic agents.

10. The method of claim 9, wherein the at least one resistance transgene encodes a polypeptide selected from the group consisting of: ABCG2, BCRP, LRP, MRP1 , MRP2, MRP3, MRP4, MRP5, MRP6, MRP8, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S-transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2, thrombopoietin receptor

F36Vmpl, IRDS (interferons), HREP450R, SIRT1 , and variants, and functional fragments thereof.

1 1 . The method of claim 1 , wherein each of the plurality of genetically modified cells comprises at least one cell suicide transgene that is capable of inducing the apoptosis of said cells.

12. The method of claim 1 1 , wherein at least one cell suicide transgene encodes a polypeptide selected from the group consisting of: caspase-3, caspase-8, caspase-9, caspase-12, apoptosis inducing factor, BAD, and BIM.

13. The method of claim 1 1 , wherein the at least one cell suicide transgene encodes a polypeptide selected from the group consisting of: thymidine kinase, HSVTK39, carboxylesterase , carboxypeptidase G2, cytochrome P450, cytosine deaminase, nitroreductase 1 , FAS-FK506 binding protein (FKBP) fusion protein, FADD-FK506 fusion protein, caspase-3 death domain-FK506 fusion protein, caspase-6 death domain-FK506 fusion protein, caspase-7 death domain-FK506 fusion protein, caspase-8 death domain-FK506 fusion protein, caspase-9 death domain-FK506 fusion protein, caspase-10 death domain-FK506 fusion protein, CD20, novel EC domains, and novel EC domains fused to chemical dimerization domain and intracellular death domain.

14. The method of claim 13, wherein apoptosis is induced by contacting the plurality of genetically modified cells with a small molecule.

15. The method of claim 14, wherein the small molecule is converted into a cytotoxic agent that induces apoptosis in the genetically modified cells.

16. The method of claim 14, wherein the small molecule allows the multimerization of polypeptides that induce apoptosis upon multimerization.

17. The method of claim 1 1 , wherein the at least one cell suicide transgene encodes an RNAi molecule directed against a gene encoding a

polypeptide that is essential for cell survival.

18. The method of claim 17 wherein the gene that encodes a polypeptide that is essential for cell survival is selected from the group consisting of: survivin, IAP, and Bcl2.

19. The method of claim 2, wherein the site in vivo is selected from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, or kidney.

20. The method of claim 1 , wherein the one or more cytotoxic agents are selected from the group consisting of: chemotherapeutic agents, alkyl sulfonates, aziridines, ethylenimines, methylamelamines, delta-9- tetrahydrocannabinol, beta-lapachone, lapachol, colchicines, betulinic acid, camptothecins, bryostatin, callystatin, CC-1065, podophyllotoxin, podophyllinic acid, teniposide, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards, nitrosureas, antibiotics, antimetabolites, folic acid analogues, purine analogs, pyrimidine analogs, androgens, anti-adrenals, maytansinoids, taxoids, platinum analogs, topoisomerase inhibitors, retinoids, aptamers, protein kinase inhibitors, and pharmaceutically acceptable salts, acids or derivatives.

21 . The method of claim 1 , wherein at least one of plurality of the genetically modified cells remains at the site for about 1 week to about 1 year.

22. The method of claim 1 , wherein at least one of plurality of the genetically modified cells remains at the site for about 1 month to about 1 year.

23. The method of claim 1 , wherein at least one of plurality of the genetically modified cells remains at the site for about 6 months to about 1 year.

24. The method of claim 1 , wherein at least one of plurality of the genetically modified cells remains at the site for about 1 year.

25. The method of claim 1 , wherein at least one of plurality of the genetically modified cells remains at the site for more than 1 year.

26. The method of any one of claim 1 , wherein each of the genetically modified cells comprises at least one factor transgene that encodes a dedifferentiation factor, a differentiation factor, a growth factor or a cytokine.

27. The method of claim 26, wherein the factor transgene encodes at least the dedifferentiation factors Oct ¾, Sox 2, and Nanog.

28. The method of claim 26, wherein the factor transgene encodes the dedifferentiation factor Oct ¾.

29. The method of claim 26, wherein the factor transgene encodes a growth factor or cytokine selected from the group consisting of: brain derived neurotropihic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (BMP-2), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 - CCL28, CXCL1 -CXCL17, XCL1 , XCL2, CX3CL1 , vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF), FAS-ligand, fibroblast growth factor 1 (FGF-1 ), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 (FGF-10), Flt-3, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM- CSF), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN- b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1 ), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 1 1 (IL-1 1 ), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL- 17), interleukin 19 (IL-19), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derived growth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF-CC), platelet derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell derived factor 1 (SDF-1 ), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), Wnt1 , Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt1 Ob, Wnt1 1 , Wnt14, Wnt15, or Wnt16, Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

30. The method of any one of claims 1 , 9, 1 1 , or 26, wherein expression of one or more transgenes is inducible.

31 . The method of any one of claims 1 , 9, 1 1 , or 26, wherein expression of one or more transgenes is constitutive.

32. The method of claim 1 , wherein a plurality of non-genetically modified cells is also administered to the individual.

33. The method of claim 32, wherein the plurality of non-genetically modified cells is selected from the group consisting of: isolated adult stem cells, isolated embryonic stem cells, induced pluripotent stem cells, and cells differentiated therefrom.

34. The method of claim 32, wherein the non-genetically modified cells are administered prior to the genetically modified cells.

35. The method of claim 32, wherein the non-genetically modified cells are administered at substantially the same time as the genetically modified cells.

36. The method of claim 32, wherein the genetically modified cells are administered prior to the non-genetically modified cells.

37. A kit comprising:

(a) one or more cytotoxic agents that target at least one population of susceptible cells;

(b) a plurality of genetically modified cells comprising:

(i) at least one resistance transgene that provides resistance to the one or more cytotoxic agents; and

(ii) at least one inducible cell suicide transgene; and

(c) a pharmaceutically acceptable cell culture medium.

38. The kit of claim 37, wherein the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell.

39. The kit of claim 37, wherein the plurality of genetically modified cells comprises a cell differentiated from an isolated adult stem cell, an isolated embryonic stem cell, or an induced pluripotent stem cell.

40. The kit of claim 37, wherein the plurality of genetically modified cells comprises a somatic cell.

41 . The kit of any one of claims 37-40, wherein the plurality of genetically modified cells is isolated from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, hematopoietic cells, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, and kidney.

42. The kit of any one of claims 37-40, wherein the plurality of genetically modified cells is mammalian.

43. The kit of any one of claims 37-40, wherein the plurality of genetically modified cells is human.

44. The kit of claim 37, comprising a plurality of non-genetically modified cells.

45. The kit of claim 44, wherein the plurality of non-genetically modified cells is selected from the group consisting of: isolated adult stem cells, isolated embryonic stem cells, induced pluripotent stem cells, and cells differentiated therefrom.

46. The kit of claim 37, wherein the at least one resistance transgene that provides resistance to one or more cytotoxic agents encodes a polypeptide selected from the group consisting of: ABCG2, BCRP, LRP, MRP1 , MRP2, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S- transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2,

thrombopoietin receptor F36Vmpl, IRDS (interferons), HREP450R, SIRT1 , and variants, and functional fragments thereof.

47. The kit of claim 37, wherein the at least one inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: caspase-3, caspase-8, caspase-9, caspase-12, apoptosis inducing factor, BAD, and BIM.

48. The kit of claim 37, wherein the at least one cell inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: thymidine kinase, HSVTK39, carboxylesterase , carboxypeptidase G2, cytochrome P450, cytosine deaminase, nitroreductase 1 , FAS-FK506 binding protein (FKBP) fusion protein, FADD-FK506 fusion protein, caspase-3 death domain-FK506 fusion protein, caspase-6 death domain-FK506 fusion protein, caspase-7 death domain- FK506 fusion protein, caspase-8 death domain-FK506 fusion protein, caspase-9 death domain-FK506 fusion protein, caspase-10 death domain-FK506 fusion protein, CD20, novel EC domains, and novel EC domains fused to chemical dimerization domain and intracellular death domain.

49. The kit of claim 48, comprising a small molecule that induces apoptosis by contacting the plurality of genetically modified cells.

50. The kit of claim 49, wherein the small molecule is converted into a cytotoxic agent that induces apoptosis in the genetically modified cells.

51 . The kit of claim 49, wherein the small molecule allows the multimerization of polypeptides that induce apoptosis upon multimerization.

52. The kit of claim 37, wherein the at least one cell suicide transgene encodes an RNAi molecule directed against a gene encoding a

polypeptide that is essential for cell survival.

53. The kit of claim 52, wherein the gene that encodes a polypeptide that is essential for cell survival is selected from the group consisting of: survivin, IAP, and Bcl2.

54. The kit of claim 37, wherein the plurality of genetically modified cells is capable of being directed to a site in vivo, wherein the site is selected from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, or kidney.

55. The kit of claim 37, wherein the one or more cytotoxic agents are selected from the group consisting of: chemotherapeutic agents, alkyl

sulfonates, aziridines, ethylenimines, methylamelamines, delta-9- tetrahydrocannabinol, beta-lapachone, lapachol, colchicines, betulinic acid, camptothecins, bryostatin, callystatin, CC-1065, podophyllotoxin, podophyllinic acid, teniposide, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards, nitrosureas, antibiotics, antimetabolites, folic acid analogues, purine analogs, pyrimidine analogs, androgens, anti-adrenals, maytansinoids, taxoids, platinum analogs, topoisomerase inhibitors, retinoids, aptamers, protein kinase inhibitors, and pharmaceutically acceptable salts, acids or derivatives.

56. The kit of claim 37, wherein each of the genetically modified cells comprises at least one factor transgene that encodes a dedifferentiation factor, a differentiation factor, a growth factor, a cytokine, or a hormone.

57. The kit of claim 37, comprising at least one dedifferentiation factor polypeptide, differentiation factor polypeptide, growth factor polypeptide, cytokine, or hormone.

58. The kit of claim 37, comprising one or more dedifferentiation agents, differentiation agents, or agents that increase cell growth and/or proliferation.

59. The kit of claim 56, wherein the factor transgene encodes a growth factor or cytokine selected from the group consisting of: brain derived neurotropihic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone

morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (BMP-2), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 - CCL28, CXCL1 -CXCL17, XCL1 , XCL2, CX3CL1 , vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF), FAS-ligand, fibroblast growth factor 1 (FGF-1 ), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 (FGF-10), Flt-3, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM- CSF), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN- b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1 ), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 1 1 (IL-1 1 ), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL- 17), interleukin 19 (IL-19), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derived growth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF-CC), platelet derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell derived factor 1 (SDF-1 ), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), Wnti , Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnti 0a, Wnti 0b, Wnti 1 , Wnti 4, Wnti 5, or Wnti 6, Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

60. The kit of claim 37 or claim 56, wherein expression of one or more transgenes is inducible.

61 . The kit of claim 37 or claim 56, wherein expression of one or more transgenes is constitutive.

62. The kit of claim 37 or claim 56, comprising a biocompatible matrix.

63. The kit of claim 37 or claim 56, comprising an implant.

64. An implant comprising;

(a) one or more cytotoxic agents;

(b) a composition comprising:

(i) a plurality of genetically modified cells comprising: one or more resistance transgenes that provide resistance to the one or more cytotoxic agents; and

one or more inducible cell suicide transgenes;

(c) a biocompatible matrix; and

(d) a pharmaceutically acceptable cell culture medium.

65. The implant of claim 64, wherein the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell.

66. The implant of claim 64, wherein the plurality of genetically modified cells comprises a cell differentiated from an isolated adult stem cell, an isolated embryonic stem cell, or an induced pluripotent stem cell.

67. The implant of claim 64, wherein the plurality of genetically modified cells comprises a somatic cell.

68. The implant of any one of claims 64-67, wherein the plurality of genetically modified cells is isolated from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, hematopoietic cells, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, and kidney.

69. The implant of any one of claims 64-67, wherein the plurality of genetically modified cells is mammalian.

70. The implant of any one of claims 64-67, wherein the plurality of genetically modified cells is human.

71 . The implant of claim 64, comprising a plurality of non-genetically modified cells.

72. The implant of claim 71 , wherein the plurality of non-genetically modified cells is selected from the group consisting of: isolated adult stem cells, isolated embryonic stem cells, induced pluripotent stem cells, and cells differentiated therefrom.

73. The implant of claim 64, wherein the at least one resistance transgene that provides resistance to one or more cytotoxic agents encodes a polypeptide selected from the group consisting of: ABCG2, BCRP, LRP, MRP1 , MRP2, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S- transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2,

74. The implant of claim 64, wherein the at least one inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: caspase-3, caspase-9, apoptosis inducing factor, BAD, and BIM.

75. The implant of claim 64, wherein the at least one cell inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: thymidine kinase, HSVTK39, carboxylesterase , carboxypeptidase G2, cytochrome P450, cytosine deaminase, nitroreductase 1 , FAS-FK506 binding protein (FKBP) fusion protein, FADD-FK506 fusion protein, caspase-3 death domain-FK506 fusion protein, caspase-6 death domain-FK506 fusion protein, caspase-7 death domain- FK506 fusion protein, caspase-8 death domain-FK506 fusion protein, caspase-9 death domain-FK506 fusion protein, caspase-10 death domain-FK506 fusion protein, CD20, novel EC domains, and novel EC domains fused to chemical dimerization domain and intracellular death domain.

76. The implant of claim 75, wherein apoptosis is induced by contacting the plurality of genetically modified cells with a small molecule.

77. The implant of claim 76, wherein the small molecule is

converted into a cytotoxic agent that induces apoptosis in the genetically modified cells.

78. The implant of claim 76, wherein the small molecule allows the multimerization of polypeptides that induce apoptosis upon multimerization.

79. The implant of claim 64, wherein the at least one cell suicide transgene encodes an RNAi molecule directed against a gene encoding a

polypeptide that is essential for cell survival.

80. The implant of claim 64, wherein the gene that encodes a polypeptide that is essential for cell survival is selected from the group consisting survivin, IAP, and Bcl2.

81 . The implant of claim 64, wherein the one or more cytotoxic agents are selected from the group consisting of: chemotherapeutic agents, alkyl sulfonates, aziridines, ethylenimines, methylamelamines, delta-9- tetrahydrocannabinol, beta-lapachone, lapachol, colchicines, betulinic acid, camptothecins, bryostatin, callystatin, CC-1065, podophyllotoxin, podophyllinic acid, teniposide, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards, nitrosureas, antibiotics, antimetabolites, folic acid analogues, purine analogs, pyrimidine analogs, androgens, anti-adrenals, maytansinoids, taxoids, platinum analogs, topoisomerase inhibitors, retinoids, aptamers, protein kinase inhibitors, and pharmaceutically acceptable salts, acids or derivatives.

82. The implant of claim 64, wherein each of the genetically modified cells comprises at least one factor transgene that encodes a

dedifferentiation factor, a differentiation factor, a growth factor, a cytokine, or a hormone.

83. The implant of claim 64, comprising at least one dedifferentiation factor polypeptide, differentiation factor polypeptide, growth factor polypeptide, cytokine, or hormone.

84. The implant of claim 64, comprising one or more

85. The implant of claim 82, wherein the factor transgene encodes a growth factor or cytokine selected from the group consisting of: brain derived neurotropihic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (BMP-2), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 - CCL28, CXCL1 -CXCL17, XCL1 , XCL2, CX3CL1 , vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF), FAS-ligand, fibroblast growth factor 1 (FGF-1 ), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 (FGF-10), Flt-3, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM- CSF), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN- b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1 ), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 1 1 (IL-1 1 ), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL- 17), interleukin 19 (IL-19), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derived growth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF-CC), platelet derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell derived factor 1 (SDF-1 ), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), Wnti , Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnti 0a, Wnti 0b, Wnti 1 , Wnti 4, Wnti 5, or Wnti 6, Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

86. The implant of claim 64, wherein the biocompatible matrix comprises one or more components selected from the group consisting of: a polymer, a biologically inert particle, a polypeptide, an oligosaccharide, a lipid, a polynucleotide, or a small molecule or any combination thereof

87. The implant of claim 86, wherein the polymer comprises one or more of polylactic acid, polyglycolic acid, polycaprolactone, polylactic acid- polyglycolic acid copolymer, polylactic acid-polycaprolactone copolymer, and polyglycolic acid-polycaprolactone copolymer.

88. The implant of claim 86, wherein the biologically inert particle is selected from the group consisting of: a magnetic particle, an agarose particle, and a plastic particle.

89. The implant of claim 86, wherein the polypeptide is selected from the group consisting of: an antibody or antigen binding fragment thereof, a peptidomimetic, a lipoprotein, a ligand, a lectin, an Fc domain, and a cell-surface receptor or an extracellular fragment thereof.

90. The implant of claim 86, wherein the polynucleotide is selected from the group consisting of: an aptamer, a single-stranded RNA, a double-stranded RNA, a hypomethylated single-stranded DNA molecule, and synthetic analogs thereof.

91 . The implant of claim 86, wherein the small molecule is selected from the group consisting of: folate, biotin, digoxigenin, and dinitrophenyl.

92. A composition comprising:

(b) a plurality of genetically modified cells comprising:

(i) at least one resistance transgene that provides resistance to the one or more cytotoxic agents;

(ii) at least one inducible cell suicide transgene; and

(c) a pharmaceutically acceptable cell culture medium.

93. The composition of claim 92, wherein the plurality of genetically modified cells comprises at least one cell selected from the group consisting of: an isolated adult stem cell, an isolated embryonic stem cell, and an induced pluripotent stem cell.

94. The composition of claim 92, wherein the plurality of genetically modified cells comprises a cell differentiated from an isolated adult stem cell, an isolated embryonic stem cell, or an induced pluripotent stem cell.

95. The composition of claim 92, wherein the plurality of genetically modified cells comprises a somatic cell.

96. The composition of any one of claims 92-95, wherein the plurality of genetically modified cells is isolated from the group consisting of:

pancreas, CNS, PNS, heart, bone marrow, hematopoietic cells, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, and kidney.

97. The composition of any one of claims 92-95, wherein the plurality of genetically modified cells is mammalian.

98. The composition of any one of claims 92-95, wherein the plurality of genetically modified cells is human.

99. The composition of claim 92, comprising a plurality of non- genetically modified cells.

100. The composition of claim 99, wherein the plurality of non- genetically modified cells is selected from the group consisting of: isolated adult stem cells, isolated embryonic stem cells, induced pluripotent stem cells, and cells differentiated therefrom.

101 . The composition of claim 92, wherein the at least one resistance transgene that provides resistance to one or more cytotoxic agents encodes a polypeptide selected from the group consisting of: ABCG2, BCRP, LRP, MRP1 , MRP2, MDR1 , MGMT, glycosylases, aldehyde dehydrogenase, glutathione S- transferase, superoxide dismutase 2, dihydrofolate reductase, thymidylate synthase, cytosine deaminase, surviving, inhibitor of apoptosis peptide (IAP), Bcl2,

102. The composition of claim 92, wherein the at least one inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: caspase-3, caspase-9, apoptosis inducing factor, BAD, and BIM.

103. The composition of claim 92, wherein the at least one cell inducible cell suicide transgene encodes a polypeptide selected from the group consisting of: thymidine kinase, HSVTK39, carboxylesterase , carboxypeptidase G2, cytochrome P450, cytosine deaminase, nitroreductase 1 , FAS-FK506 binding protein (FKBP) fusion protein, FADD-FK506 fusion protein, caspase-3 death domain-FK506 fusion protein, caspase-6 death domain-FK506 fusion protein, caspase-7 death domain-FK506 fusion protein, caspase-8 death domain-FK506 fusion protein, caspase-9 death domain-FK506 fusion protein, caspase-10 death domain-FK506 fusion protein, CD20, novel EC domains, and novel EC domains fused to chemical dimerization domain and intracellular death domain.

104. The composition of claim 103, wherein apoptosis is induced by contacting the plurality of genetically modified cells with a small molecule.

105. The composition of claim 103, wherein the small molecule is converted into a cytotoxic agent that induces apoptosis in the genetically modified cells.

106. The composition of claim 103, wherein the small molecule allows the multimerization of polypeptides that induce apoptosis upon

multimerization.

107. The composition of claim 92, wherein the at least one cell suicide transgene encodes an RNAi molecule directed against a gene encoding a polypeptide that is essential for cell survival.

108. The composition of claim 107, wherein the gene that encodes a polypeptide that is essential for cell survival is selected from the group consisting of: survivin, IAP, and Bcl2.

109. The composition of claim 92, wherein the plurality of genetically modified cells is capable of being directed to a site in vivo, wherein the site is selected from the group consisting of: pancreas, CNS, PNS, heart, bone marrow, blood, umbilical cord, skeletal muscle, smooth muscle, bone, skin, liver, hair follicles, vascular system, adipose, lung, retina, cornea, or kidney.

1 10. The composition of claim 92, wherein the one or more cytotoxic agents are selected from the group consisting of: chemotherapeutic agents, alkyl sulfonates, aziridines, ethylenimines, methylamelamines, delta-9- tetrahydrocannabinol, beta-lapachone, lapachol, colchicines, betulinic acid, camptothecins, bryostatin, callystatin, CC-1065, podophyllotoxin, podophyllinic acid, teniposide, cryptophycins, dolastatin, duocarmycin, eleutherobin, pancratistatin, sarcodictyin, spongistatin, nitrogen mustards, nitrosureas, antibiotics, antimetabolites, folic acid analogues, purine analogs, pyrimidine analogs, androgens, anti-adrenals, maytansinoids, taxoids, platinum analogs, topoisomerase inhibitors, retinoids, aptamers, protein kinase inhibitors, and pharmaceutically acceptable salts, acids or derivatives.

1 1 1 . The composition of claim 92, wherein each of the genetically modified cells comprises at least one factor transgene that encodes a

1 12. The composition of claim 92, comprising at least one dedifferentiation factor polypeptide, differentiation factor polypeptide, growth factor polypeptide, cytokine, or hormone.

1 13. The composition of claim 92, comprising one or more dedifferentiation agents, differentiation agents, or agents that increase cell growth and/or proliferation.

1 14. The composition of claim 1 1 1 , wherein the factor transgene encodes a growth factor or cytokine selected from the group consisting of: brain derived neurotropihic factor (BDNF), bone morphogenetic protein 2 (BMP-2), bone morphogenetic protein 6 (BMP-6), bone morphogenetic protein 7 (BMP-7), cardiotrophin 1 (BMP-2), CD22, CD40, ciliary neurotrophic factor (CNTF), CCL1 - CCL28, CXCL1 -CXCL17, XCL1 , XCL2, CX3CL1 , vascular endothelial cell growth factor (VEGF), epidermal growth factor (EGF), FAS-ligand, fibroblast growth factor 1 (FGF-1 ), fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), fibroblast growth factor 5 (FGF-5), fibroblast growth factor 6 (FGF-6), fibroblast growth factor 1 (FGF-7), fibroblast growth factor 1 (FGF-10), Flt-3, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage stimulating factor (GM- CSF), hepatocyte growth factor (HGF), interferon alpha (IFN-a), interferon beta (IFN- b), interferon gamma (IFNg), insulin-like growth factor 1 (IGF-1 ), insulin-like growth factor 2 (IGF-2), interleukin 1 (IL-1 ), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 9 (IL-9), interleukin 10 (IL-10), interleukin 1 1 (IL-1 1 ), interleukin 12 (IL-12), interleukin 13 (IL-13), interleukin 15 (IL-15), interleukin 17 (IL- 17), interleukin 19 (IL-19), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein 1 (MCP-1 ), macrophage inflammatory protein 3a (MIP-3a), macrophage inflammatory protein 3b (MIP-3b), nerve growth factor (NGF), neurotrophin 3 (NT-3), neurotrophin 4 (NT-4), platelet derived growth factor AA (PDGF-AA), platelet derived growth factor AB (PDGF-AB), platelet derived growth factor BB (PDGF-BB), platelet derived growth factor CC (PDGF-CC), platelet derived growth factor DD (PDGF-DD), RANTES, stem cell factor (SCF), stromal cell derived factor 1 (SDF-1 ), transforming growth factor alpha (TGF-a), transforming growth factor beta (TGF-b), tumor necrosis factor alpha (TNF-a), Wnt1 , Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt1 Ob, Wnt1 1 , Wnt14, Wnt15, or Wnt16, Sonic hedgehog, Desert hedgehog, and Indian hedgehog.

1 15. The composition of claim 92 or claim 1 1 1 , wherein expression of one or more transgenes is inducible.

1 16. The composition of claim 92 or claim 1 1 1 , wherein expression of more transgenes is constitutive.