WO2000006204A2 - Cellules genetiquement modifiees et tissus - Google Patents

Cellules genetiquement modifiees et tissus Download PDF

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Publication number
WO2000006204A2
WO2000006204A2 PCT/US1999/016961 US9916961W WO0006204A2 WO 2000006204 A2 WO2000006204 A2 WO 2000006204A2 US 9916961 W US9916961 W US 9916961W WO 0006204 A2 WO0006204 A2 WO 0006204A2
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Prior art keywords
cells
gene
composition
tissues
expression
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PCT/US1999/016961
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English (en)
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WO2000006204A3 (fr
Inventor
Gail K. Naughton
Jonathon N. Mansbridge
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Advanced Tissue Sciences, Inc.
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Publication date
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Priority to KR1020017001189A priority Critical patent/KR20010085313A/ko
Priority to EP99938808A priority patent/EP1100548A2/fr
Priority to AU53212/99A priority patent/AU5321299A/en
Priority to JP2000562057A priority patent/JP2002521046A/ja
Priority to CA002338487A priority patent/CA2338487A1/fr
Publication of WO2000006204A2 publication Critical patent/WO2000006204A2/fr
Publication of WO2000006204A3 publication Critical patent/WO2000006204A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to cells genetically engineered to express a target gene in response to a given "environmental cue” or "stimulus”, and their use in generating three-dimensional tissues having improved properties for production and processing, or superior properties for use in vivo, in tissue or organ repair or as bioreactors for the delivery of therapeutic products.
  • the majority of vertebrate cell cultures in vitro are grown as monolayers on an artificial substrate bathed in nutrient medium.
  • the nature of the substrate on which the monolayers grow may be solid, such as plastic, or semisolid gels, such as collagen or agar. Disposable plastics have become the preferred substrate used in modern-day tissue or cell culture.
  • Basement membranes comprise a mixture of glycoproteins and proteoglycans that surround most cells in vivo.
  • Reid and Rojkund, 1979 in, Methods in Enzymology, Vol. 57, Cell Culture, Jakoby & Pasten. eds., New York, Acad. Press, pp. 263-278; Vlodavsky et al., 1980, Cell 19:607-617; Yang et al., 1979, Proc. Natl. Acad. Sci. USA 76:3401 have used collagen for culturing hepatocytes, epithelial cells and endothelial tissue.
  • mice embryo fibroblasts have been used to enhance the growth of cells, particularly at low densities. This effect is thought to be due partly to supplementation of the medium but may also be due to conditioning of the substrate by cell products.
  • feeder layers of fibroblasts are grown as confluent monolayers which make the surface suitable for the attachment of other cells. For example, the growth of glioma cells on confluent feeder layers of normal fetal intestine has been reported (Lindsay. 1979. Nature 228:80).
  • these three-dimensional substrates are inoculated with the cells to be 5 cultured.
  • Many of the cell types have been reported to penetrate the matrix and to establish a "tissue-like" histology.
  • tissue-like substrates have been utilized to culture breast epithelium (Yang et al., 1981, Cancer Res. 41 : 1021- 1027) and sympathetic neurons (Ebendal, 1976, Exp. Cell Res. 98:159-169). Additionally, various attempts have been made to regenerate tissue-like architecture
  • a hydrated bovine collagen lattice forms the substrate to which cells, such as fibroblasts are incorporated which results in the contraction of the lattice into tissue (Bell et al., U.S. Patent No. 4,485,096).
  • a porous cross-linked collagen sponge is used to culture fibroblast cells (Eisenberg, WO 91/16010).
  • a scaffold composed of synthetic polymers has also been i Q described to control cell growth and proliferation in vitro so that once the fibroblasts begin to grow and attach to the matrix it is transplanted into the patient (Vacanti et al., U.S. Patent Nos. 5,759,830; 5,770,193; 5,736,372).
  • Additional synthetic matrices composed of biodegradable, biocompatible copolymers of polyesters and amino acids have also been designed as scaffolding for cell growth (U.S. Patent Nos. 5,654.381 ;
  • Three-dimensional cell culture systems have also been designed which are composed of a stromal matrix which supports the growth of cells from any desired tissue into an adult tissue (Naughton et al, U.S. Patent Nos. 4,721,096, 4,963.489 and 5,032,508). Another approach involves slowly polymerizing hydrogels containing large numbers of the desired cell type which harden into a matrix once administered to 0 a patient (U.S. Patent No. 5,709,854). Extracellular matrix preparations have been designed which are composed of stromal cells which provide a three dimensional cell culture system for a desired cell type which may be injected into the patient for precise placement of the biomaterial (Naughton et al., WO 96/39101).
  • Gene therapy protocols are directed to the delivery of biologically active gene products or the delivery of replacement genes to correct heritable defects in target cells.
  • ex vivo gene transfer and in vivo gene transfer.
  • Any number of transfection techniques which are used to transfer DNA in vitro into cells may be used for ex vivo gene transfer, including calcium phosphate - DNA precipitation; DEAE-Dextran transfection. electroporation, Hposome mediated DNA transfer or transduction with recombinant viral vectors. These transfection techniques have been used to transfer DNA into a variety of cell types, including endothelial cells.
  • hepatocytes e.g., see U.S. Patent No. 4,868,1 16, U.S. Patent No. 4.980,286; Morgan & Mulligan WO 87/00201 ; WO 89/07136; WO 89/00201 ; WO 89/07136; U.S. Patent No. 4,963,489; U.S. Patent No. 5,399,346).
  • Viral vectors are regarded as the most efficient system, and recombinant replication-defective viral vectors have been used to transduce (i.e., infect) cells both ex vivo and in vivo.
  • Such vectors have included retroviral, adenovirus and adeno- associated and herpes viral vectors.
  • viral vectors While highly efficient at gene transfer, the major disadvantages associated with the use of viral vectors include the inability of many viral vectors to infect non-dividing cells; problems associated with insertional mutagenesis; inflammatory reactions to the virus and potential helper virus production, and/or production and transmission of harmful virus to other human patients.
  • the present invention relates to cells genetically engineered to express a target gene in response to a given "environmental cue” or "stimulus,” and their use for the production of three-dimensional tissues or injectable preparations which can be used in tissue repair, replacement or enhancement, and/or for the delivery of therapeutic gene products in vivo.
  • the cells used in the invention are genetically engineered to express a desired target gene in response to induction by a substance naturally produced by the subject/recipient of the cells, or by a substance administered to the subject
  • the genetically engineered cells and tissues of the invention can be designed to deliver desired gene products locally (at the site of implantation) or systemically
  • the target gene product may be therapeutic or a detectable gene product that functions as an indicator for the subject - !_£_, indicating that medication or medical attention is required
  • the genetically engineered cells and tissues of the invention can be used not only to deliver gene products in vivo but also to improve the properties (i e , the production, processing, storage and/or use by the physician) of the cells and tissues destined for implantation in vivo, and most importantly, in human subjects
  • the cells and tissues of the present invention are engineered to express a first gene product that responds to a given "environmental cue” or “stimulus,” and m response to the "cue” or “stimulus” directly or indirectly regulates the expression of the target gene
  • the cells and tissues of the invention can be genetically engineered to express a receptor that responds to a physiological change in the subject recipient a ligand, and/or to a compound administered to the subject/recipient (e g , endogenous factors or hormones produced temporally by subject, exogenous factors or compounds administered to the patient to induce expression of the gene product; and physical changes or stresses such as heat shock )
  • the response of the receptor in turn, induces the expression of a target gene
  • the target gene which may be engineered into the cell or endogenous, is controlled by a regulatory sequence (e g , a promoter, promoter element and/or enhancer) that is responsive to activation of the first gene product, e g , the receptor
  • cells genetically engineered to express a target gene in response to an "environmental cue” or "stimulus” are further engineered to generate tissues having superior properties for use in vivo.
  • tissues having superior properties include, but are not limited to, engineered tissues that express gene products that reduce the inflammatory and/or immune response at the site of implantation; engineered cells which express gene products that render the tissue resistant to infection by certain pathogens; engineered cells which express gene products that modulate angiogenesis to provide the appropriate vascularization for the implanted tissues; engineered cells which express gene products with improved wound-healing properties; engineered cells which express gene products that have anti-scarring properties; and engineered cells which express a cryoprotectant or anti-desiccant to impart the product with improved storage properties for freezing, drying or freeze-drying.
  • the three-dimensional tissues can be prepared using genetically engineered stromal cells, parenchymal cells, or both. Techniques for achieving improved transfection efficiencies are described.
  • cells which normally display limited passages in culture can be reversibly immortalized to allow for more efficient engineering — in this embodiment, normal cells (which can be derived from the patient or subject) can be reversibly immortalized by genetically engineering the telomerase gene into the cells.
  • the immortalized cells can be expanded, cloned and more efficiently engineered.
  • the genetically engineered cells can then be converted back to non-immortalized cells which are more suitable for use in vivo by removing or disrupting the telomerase gene.
  • the genetically engineered cells can be designed to contain, in addition to the gene of interest, a "suicide" gene that can be induced in the event the implanted cells or tissue need to be shut down or removed from the body.
  • the genetically engineered cells and tissues of the present invention have a wide array of applications, ranging from gene therapy, including transplantation or implantation in vivo, to screening cytotoxic compounds or pharmaceutical compounds in vivo.
  • the engineered cells and tissues may be used to test the cytotoxicity of vectors and other nucleic acids and their encoded gene products in gene therapy applications
  • Adherent Layer cells attached directly to the three dimensional framework or connected indirectly by attachment to cells that are themselves attached directly to the framework , o Stromal Cells fibroblasts with or without other cells and/or elements found in loose connective tissue, including but not limited to, endothelial cells, pe ⁇ cytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, etc
  • Tissue-Specific or Parenchymal Cells the cells which form the essential and distinctive tissue of an organ as distinguished from its supportive stromal mat ⁇ x 15
  • Three-Dimensional Framework- a three dimensional framework composed of any matenal and/or shape that (a) allows cells to attach to it (or can be modified to allow cells to attach to it), and (b) allows cells to grow in more than one layer This support is inoculated with stromal cells to form the three-dimensional stromal mat ⁇ x.
  • Three-Dimensional Stromal Matnx a three dimensional framework which has -j Q been inoculated with stromal cells Whether confluent or subconfluent, stromal cells according to the invention continue to grow, divide and elaborate extracellular mat ⁇ x proteins. The stromal matrix will support the growth of tissue — specific cells later inoculated to form the three dimensional cell culture
  • tissue specific cells used to inoculate the three-dimensional stromal mat ⁇ x should include the "stem” cells (or “reserve” cells) for that tissue; I ⁇ , those cells which generate new cells that will mature into the specialized cells that form the parenchyma of the tissue.
  • CFU-GEMM colony forming unit-granuloid, erythroid, monocyte. megakaryocyte
  • EDTA ethylene diamine tetraacetic acid
  • LTBMC long term bone marrow culture
  • PBL pe ⁇ pheral blood leukocytes
  • PBS phosphate buffered saline
  • RPMI 1640 Rosewell Park Memo ⁇ al Institute medium number 1640 (GIBCO, Inc., Grand Island, NY)
  • FIG. 1 is a scanning electron micrograph depicting fibroblast attachment to the three-dimensional framework and extension of cellular processes across the mesh opening. Fibroblasts are actively secreting mat ⁇ x proteins and are at the approp ⁇ ate stage of subconfluency which should be obtained prior to inoculation with tissue- specific cells.
  • FIG. 2 is a scanning electron micrograph of the three-dimensional long term bone marrow culture (LTBMC) demonstrating the 210 ⁇ m sieve area for expression of erythroid, myeloid and other colonies. Support cells have grown linearly along and enveloped .he three-dimensional framework.
  • LTBMC long term bone marrow culture
  • FIG. 3 is diagrammatic representation of the three-dimensional skin model.
  • a dermal/epidermal junction is present, above which lies pigmented melanocytes and several layers of pigment-containing keratinocvtes.
  • the stromal cells attach to the framework and form the dermal component.
  • FIG. 4 is a scanning electron micrograph of the three-dimensional stroma three days after inoculation with melanocytes. Melanocytes grow normally in the three- dimensional system in that they exhibit dend ⁇ te formation, remain pigmented. and retain the ability to transfer pigment to keratinocytes.
  • FIG. 5 is a photomicrograph of the three-dimensional skin model grafted onto rats seven days post transplant. A distinct dermal and epidermal junction is evident.
  • FIG. 6 is a photomicrograph of the three-dimensional skin model grafted onto rats seven days post transplant.
  • Collagen bundles (c) and all cell types are represented, including keratinocytes (k).
  • the injectable or implantable compositions of the invention are composed, in whole or in part, of cells engineered to express a first gene product(s) that responds to j 0 a preselected "environmental cue” or "stimulus".
  • the response of the first gene product directly or indirectly regulates the expression of one or more target genes.
  • the expression of the target or second gene product is driven by a promoter activated directly or indirectly in response to the first gene product's response to a "cue" or "stimulus".
  • the term "environmental cue” or j 5 "stimulus” used herein refers to any signal (e.g., endogenous factors or hormones produced temporally by the subject; exogenous factors or compounds administered to the patient to induce expression of the target gene product; and physical changes or stress such as heat shock) that a cell can be genetically engineered to respond to.
  • the "cue” or “stimulus” may be a biological molecule (e.g., a peptide, a 0 protein, an antibody, a carbohydrate, a lipid or a nucleic acid) that binds to a receptor expressed by the engineered cells or tissues and induces the activation of a signaling cascade that results in the expression of a target gene product.
  • target gene products include, but are not limited to, proteins such as enzymes, hormones, cytokines, antigens, antibodies, regulatory proteins, transcription proteins, receptors, and structural 5 proteins, or nucleic acid products such as antisense RNA or ribozymes.
  • the expression of the target or second gene product corrects, modifies or regulates the physiological condition to which the engineered cell responds.
  • the cells or tissues of the present invention may be engineered to express insulin in response to changes in the level of glucose.
  • the engineered cells can be used to generate a skin patch that supplies the target gene product in response to a stimulus that is applied topically.
  • the cells in the patch can be engineered to express a corticosteroid receptor that activates expression of a target gene controlled by a responsive regulatory element.
  • Target gene expression can be induced by topical 5 appliction of a co ⁇ icosteroid.
  • the expression of the target or second gene product e.g., a reporter gene product such as ⁇ -galactosidase or green fluorescent protein (GRP)
  • GFP green fluorescent protein
  • the genetically engineered cells can be used to generate an implantable "indicator" skin patch that responds to physiological changes in the recipient, and can be used to monitor diseases and disorders.
  • the cells in the patch can be engineered to express a receptor that responds to high glucose concentrations and induces expression of the target indicator gene.
  • the implanted skin patch can be used by the recipient to monitor blood glucose levels.
  • the cells and tissues of the present invention are genetically engineered to express multiple gene products that respond to different "environmental cues" or “stimuli", and in response, induce the expression of different target gene products.
  • the cells and tissues are genetically engineered to express target gene products under the control of different regulatory elements.
  • the cells and tissues are engineered to express a first gene product(s) that responds to a given "environmental cue” or “stimulus”, and in response to the "cue” or “stimulus” directly or indirectly regulates the expression of a second gene product, which indicates that a compound should be taken to induce the expression of a third or target gene product.
  • the cells and tissues may be engineered to constitutively express a cell surface receptor, such as CD4, for a particular virus.
  • a viral gene product e.g., tat
  • Detection of the expression of the reporter gene indicates that a compound that induces the expression of a peptide, protein or ribozyme that inhibits viral replication or enhances the immune system should be administered to the individual.
  • the term "compound” as used herein refers to a naturally occurring or synthetic compound which is physiologically acceptable, can be readily administered (i.e., ingested, injected or topically applied) and crosses the necessary membranes to induce the expression of a target gene product.
  • the compound for example, may be a pill that is ingested or an ointment that is topically applied.
  • the compound is designed to specifically induce the expression of the target gene.
  • the engineered cells and tissues are capable of regulating the expression of the target gene product in an autocrine or paracrine
  • SUBST ⁇ UTE SHEET (RULE 26) manner.
  • the benefit of enginee ⁇ ng cells or tissues that express a target gene product, which corrects, modifies, or stabilizes a physiological condition, upon receptor-ligand interaction is that the system is self-regulating — once the stimulus is removed the cells
  • the genetically engineered cells and tissues of the present invention can be used as bioreactors for the production and delivery of therapeutic and/or biologically active gene products in vivo, Le , in human or animal subjects.
  • the genetically engineered cells and tissues of the present j 0 invention are used in vitro or in vivo (in test animals) to screen for agents, such as pharmaceutical compounds or cytotoxic compounds, that modulate the expression of a target gene product directly or indirectly.
  • the genetically engineered cells and tissues of the present invention are used in vitro or in vivo to identify agents that modulate the activity of the target gene product directly or j 5 indirectly.
  • agents include, but are not limited to, nucleic acids (e.g., DNA and RNA), proteins, peptides, carbohydrates, lipids, antibodies, and small molecules.
  • nucleic acids e.g., DNA and RNA
  • proteins e.g., proteins, peptides, carbohydrates, lipids, antibodies, and small molecules.
  • the genetically engineered cells and tissues of the present invention are used scientifically to elucidate the regulation of gene expression and the signal transduction pathways activated in response to an "environmental cue" or
  • the cells are genetically engineered to ? 5 express a first gene product(s) that responds to an "environmental cue” or “stimulus", and in response induces the expression of a target gene(s).
  • first gene product refers to any gene product that is capable of responding to an "environmental cue” or “stimulus”, and in response inducing the expression of a target gene product.
  • the first gene product may be encoded by a naturally occurring or non- 20 naturally occurring gene product.
  • non-naturally occurring polynucleotide sequence refers to any polynucleotide sequence that is not found in nature and is engineered by humans.
  • the first gene product is overexpressed, is a fusion protein, 25 and or a chimeric protein.
  • the first gene product(s) may or may not be expressed by cells normally. In a preferred embodiment, the first gene product(s) is not expressed by the cells normally.
  • the expression of the first gene product is controlled by constitutive or tissue- specific regulatory elements.
  • constitutive regulator y elements including but not limited to: the SV40 early promoter region (Bemoist and Chambon, 1981, Nature 290, 304-310), the promoter contained in the 3 ' long terminal repeat of Rous sarcoma virus (Yamamoto. et al., 1980, Cell 22, 787-797), and the herpes thymidine kinase promoter (Wagner, et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 1441-1445).
  • transcriptional control regions that exhibit tissue specificity that may be utilized to regulate expression of a first gene, include but are not limited to: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, cell 38:639—646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Bid.
  • tissue-specific tyrosinase promoter elements (Diaz et al., 1998, J. 0 Virol. 72: 789); muscle specific promoter elements (Baker et al., 1998, Nucleic Acids Res. 26:1092), prostate-specific enhancer elements (Schuur et al., 1996, J. Biol. Chem. 271 : 7043), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1 :268 — 276); alphafetoprotein gene control region which is active in liver (Krumlauf et al.. 1985. Mol. Cell. Biol.
  • alpha — 1 antitrypsin gene control region which is active in liver (Kelsey et al., 1987, Genes and Devel.
  • beta — globin gene control region which is active in myeloid cells (Magram et al., 1985, Nature 315:338 — 340; Kollias et al, 1986, Cell 46:89 — 94); myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703 — 712); myosin 0 light chain — 2 gene control region which is active in skeletal muscle (Shani, 1985, Nature 314:283-286); and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).
  • the first gene product is a receptor.
  • receptor refers to any biological molecule(s) that is capable of specifically 5 interacting with another biological molecule, and in response directly or indirectly activates the transcription of specifically responsive genes. A functional receptor of the
  • SUBST ⁇ UTE SHEET (RULE 26) present invention may be comp ⁇ sed of one or more subumts.
  • the term "functional receptor" as used herein refers to a receptor that is capable of directly or indirectly inducing the expression of a target gene product in response to receptor- gand interaction Examples of the receptors of the present invention include, but are not limited to.
  • intracellular receptors such as steroid hormone receptors and cell surface receptors such as CD4, ⁇ nterleuk ⁇ n-2 receptor (IL-2R), ⁇ nterleukm-3 receptor (IL-3R), ⁇ nterleuk ⁇ n-4 receptor (IL-4R), mterferon receptor (IFN-R), platelet-de ⁇ ved growth factor receptor (PDGF-R), and epidermal growth factor receptor (EGF-R).
  • IL-2R ⁇ nterleuk ⁇ n-2 receptor
  • IL-3R ⁇ nterleukm-3 receptor
  • IL-4R ⁇ nterleuk ⁇ n-4 receptor
  • IFN-R mterferon receptor
  • PDGF-R platelet-de ⁇ ved growth factor receptor
  • EGF-R epidermal growth factor receptor
  • the receptor can be encoded by a naturally occur ⁇ ng polynucleotide sequence or a non- naturally occur ⁇ ng polynucleotide sequence
  • naturally occur ⁇ ng polynucleotide sequence refers to a polynucleotide sequence that is found in nature, e.g., polynucleotides found in animals, unicellular organisms, microorganisms, and viruses.
  • non-naturally occur ⁇ ng polynucleotide sequence refers to any polynucleotide sequence that is not found in nature and is engineered by humans.
  • cells and tissues are engineered to constitutively express a receptor that interacts with a particular ligand(s), and as a consequence of the receptor-ligand interaction, the expression of one or more target genes is directly or indirectly induced.
  • the expression of the target gene is driven by a promoter that is activated directly or indirectly in response to receptor-ligand interaction.
  • ligand refers to any molecule including, but not limited to, biological molecules (e g , a peptide, a protein, a carbohydrate, lipid or nucleic acid) that interacts with a receptor
  • the receptor may or may not be expressed normally by the cells that are genetically engineered. In a preferred embodiment, the receptor is not expressed normally by the cells that are genetically engineered.
  • the cells and tissues of the present invention are engineered to overexpress a receptor that interacts with a particular hgand(s), and as a consequence of the receptor-ligand interaction, expression of one or more target genes is directly or indirectly induced.
  • the overexpressed receptor may or may not expressed normally by the cells that are genetically engineered, the receptor is not expressed normally by the cells that are genetically engineered.
  • the cells and tissues overexpressing a receptor that responds to a given "environment cue" or "stimulus" are more sensitn e to the presence of a pa ⁇ icular hgand(s).
  • hormone response elements HREs
  • EGFR epidermal growth factor receptor
  • a target gene controlled by a promoter activated by the MAP kinase pathway eg , c-jun
  • receptor-ligand interaction e g , EGFR-EGF interaction
  • cells and tissues are engineered j o to express a chime ⁇ c receptor comp ⁇ sing a ligand or compound binding domain and an activation or signaling domain
  • cells and tissues of the present invention can be genetically engineered to express a chime ⁇ c receptor comp ⁇ sing a ligand or compound binding domain and a DNA binding domain
  • cells and tissues of the present invention can be genetically engineered to express a chime ⁇ c j 5 receptor comp ⁇ sing the extracellular domain of a receptor that binds to a specific ligand, a transmembrane domain of the same or a different receptor, and an intracellular domain comp ⁇ sing a signaling molecule
  • cells or tissues of the present invention are engineered express a chime ⁇ c receptor, comp ⁇ sing the extracellular domain of a receptor that binds to a specific ligand, a transmembrane 0 domain of the same or a different receptor, and the intracellular domain of a
  • cells or tissues can be engineered to express a chimeric receptor comprising an extracellular domain that binds to a specific ligand, a transmembrane domain of a receptor, and a "universal intracellular domain".
  • the term "universal intracellular domain” as used herein refers to an intracellular domain that is capable of activating the same, specific signal transduction pathway in response to receptor-ligand interaction, regardless of the ligand.
  • the "universal intracellular domain” is designed for optimal regulation of the transcription of a target gene.
  • the "universal intracellular domain" of the chimeric receptor may be comprised of the entire intracellular domain of the EpoR or a portion of it.
  • Chimeric receptors comprising, the extracellular domain of IL-3R or IL- 2R subunits and the intracellular domain of the EpoR will activate the same, specific signal transduction pathway in response to IL-3 or IL-2.
  • the promoter of a target gene is designed so that it is activated in response to ligand-chimeric receptor interaction.
  • the expression of a second or target gene product is controlled by the expression of different subunits of a receptor.
  • the sensitivity of receptor for a ligand may be regulated by expressing the high, intermediate or low affinity forms of the receptor.
  • the sensitivity of the IL-2R for IL-2 can be regulated by expressing the high affinity, heterotrimeric form ( ⁇ subunits) or the intermediate affinity, dimeric form ( ⁇ subunits) of the receptor.
  • the expression of a target gene product is controlled by modifying the expression of a receptor that specifically responds to a ligand of interest.
  • modifications that affect the sensitivity and/or specificity of a receptor for its ligand will affect the duration and quantity of the signaling molecules activated in response to ligand-receptor interaction, and thereby affect the level of expression of a target gene.
  • the sensitivity and specificity of a receptor of the present invention for a ligand is modified by altering the polynucleotide sequence encoding the receptor. Examples of such alterations to the polynucleotide sequence include additions, deletions and substitutions.
  • cells and tissues may be engineered to express an EpoR mutant (e.g., Epo-R G6002A; see Sokol, L. et al., 1995, Blood 86: 15-22) with increased sensitivity to Epo.
  • the sensitivity of a receptor of the present invention for a ligand is modified by altering the promoter that drives the expression of the receptor.
  • Engineered cells or tissues expressing high levels of a receptor of the present invention will be more sensitive to its ligand. In contrast, engineered cells or tissues expressing low levels of a receptor of the present invention will be less sensitive to its ligand.
  • the sensitivity and/or specificity of a receptor for a ligand is modified by altering the polynucleotide sequence encoding the receptor and its promoter.
  • the expression of a target gene is controlled by regulating the signal transduction pathway activated in response to receptor-ligand interaction.
  • the j o signal transduction pathway activated in response to ligand-receptor interaction may be modified by altering the polynucleotide sequence encoding the receptor.
  • cells or tissues can be engineered to express an IL-2R ⁇ subunit with the serine-region deleted. This region of the IL-2R ⁇ has been shown to be necessary for Lck activation in response to IL-2 stimulation.
  • cells or tissues expressing j the serine-region deleted IL-2R ⁇ would not induce downstream signaling molecules that require Lck for activation, e.g., MAPK.
  • the expression of a target gene is controlled by regulating the intensity and the duration of the signal transduction pathway activated in response to ligand-receptor interaction.
  • the intensity and the duration of the signal transduction pathway activated in response to ligand-receptor interaction may be modified by altering the polynucleotide sequence encoding the receptor.
  • the polynucleotide sequence encoding a receptor of the present invention may be modified to eliminate the binding site of a biological molecule that inhibits or down-regulates the 5 signal transduced in response to ligand-receptor interaction.
  • cells or tissues may be engineered to express a mutant c-Kit receptor (e.g., c-Kit with the mutationY569F; Kozlowski, M. et al, 1998, Mol Cell. Bio. 18(4):2089-2099).
  • the SH2 domain-containing SHP-1 tyrosine phosphatase which negatively regulates numerous mitogenic signaling pathways, has been shown to bind to tyrosine residues
  • the expression of a target gene is induced in response to the expression or activation of the first gene product, in response to an "environmental cue” or "stimulus".
  • the target gene product may be a peptide or protein, such as an enzyme, hormone, cytokine, antigen, or antibody, a regulatory protein, such as a transc ⁇ ption factor or DNA binding protein, a structural protein, such as a cell surface protein, or the target gene product may be a nucleic acid such as a ribosome or antisense molecule.
  • the cells and tissues of the present invention may be genetically engineered to express the following target genes:
  • Fibroblast growth factors and de ⁇ vatives thereof including FGF-2 and bFGF (U.S. Patent No. 5,026,839); FGF-1 and Cys-modified FGF (U.S. Patent No.
  • IGF-1 Human Insulin-like Growth Factor I (U.S. Patent Nos. 4,963,665 and 5,070,075);
  • EPO Erythropoietin
  • preproinsulin U.S. Patent No. 4,431,740
  • insulin analogs U.S. Patent Nos. 4,701,440, 5,008,241, 5,149,777, 5,164,366, 5,324,641, 5,618,913, 5,716,927)
  • superactive insulin analogs 4,992,417)
  • insulin precursor analogs U.S. Patent No. 5,324,641
  • the target gene product is encoded by naturally occurring polynucleotide sequences, e.g., polynucleotide sequences found in microorganisms. unicellular organisms, viruses, and animals, preferably humans.
  • the target gene product is encoded by non-naturally occurring polynucleotide sequences and is specifically designed to be stable in vivo
  • the term "non-naturally occur ⁇ ng polynucleotide sequences" encompasses polynucleotide sequences found in nature which have been modified (/ e , mutated polynucleotide sequences ⁇ having deleted sequences, additions to the sequence, and/or substitutions in the sequence)
  • a target gene(s) encodes a fusion prote ⁇ n(s) and/or chime ⁇ c prote ⁇ n(s)
  • the cells and tissues are genetically engineered to express target genes coding for gene products which have a therapeutic effect, e g , differentiation factors, growth factors, hormones, receptors and enzymes.
  • the cells and tissues are genetically engineered to express target genes coding for gene products that have an inhibitory effect, e g , ⁇ bozyme, antisense molecules and t ⁇ ple helices
  • the target gene encodes a detectable indicator gene product
  • Expression of the indicator gene product can be used to alert the subject that medical attention, or administration of the medicine is required
  • the regulatory elements operably linked to the target gene(s) bind to transc ⁇ ption factors activated, directly or indirectly, in response to the expression or activation of a first gene product(s)
  • Examples of regulatory promoters induced in response to a specific stimuli, in this the expression of a first gene product include tetracychne responsive elements, nicotine responsive elements, insulin responsive element, glucose responsive elements, interferon responsive elements, glucocorticoid responsive elements estrogen/progesterone responsive elements, retinoid acid responsive elements, viral transactivators, early or late promoter of S V40 adenovirus, the lac system, the tip system, the TAC system, the TRC system, the promoter for 3- phosphoglycerate and the promoters of acid phosphatase, and the regulatory sequences of the metallothionein gene (B ⁇ nster, et al , 1982, Nature 296, 39-42)
  • artificial response elements could be constructed, composed of multimers of transc ⁇ ption factor binding sites and hormone-res
  • cells genetically engineered to express first gene products and target gene products are further engineered to express gene products which provide enhanced properties to the genetically engineered cells.
  • cells are engineered to further express gene products which enhance cell growth, e.g., fibroblast growth factors (FGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), and transforming growth factor (TGF).
  • FGF fibroblast growth factors
  • PDGF platelet derived growth factor
  • EGF epidermal growth factor
  • TGF transforming growth factor
  • the cells and tissues are genetically engineered to further express gene products which alter the differentiation pathway of the cells.
  • cells in particular stem cells, are engineered to express intracellular or secreted proteins, e.g., DNA binding proteins, transcription factors, growth factors, hormones, cytokines, which mediate cellular interactions which influence the differentiation pathway of the cells.
  • the cells may be engineered to express a suicide gene product on cue, e.g., thymidine kinase.
  • the cells and tissues are genetically engineered to express a gene product which results in the immortalization of the cells.
  • Such an embodiment is particularly attractive for cells which normally display limited passages in culture, such as fibroblasts.
  • these cells can be reversibly immortalized to allow for more efficient engineering.
  • cells in culture or cells derived from a patient may be reversibly immortalized by genetically engineering the telomerase gene or an oncogene into the cells.
  • the immortalized cells can be expanded cloned and more efficiently engineered.
  • the genetically engineered cells can then be converted back to the non- immortalized phenotype which is more appropriate for in vivo use by disrupting or removing the telomerase gene or oncogene.
  • the telomerase gene is engineered between activatable recombination sites, such as loxP sites, which may be activated to excise the telomerase gene. Further backup safeguards may be built in to ensure that the telomerase gene is inactivated, including, but not limited to. antibiotic resistance genes and suicide genes which may be engineered into the system to ensure reliable reversible immortality.
  • the cells and tissues of the present invention are genetically engineered to further express gene products which modulate vascularization.
  • gene products which modulate vascularization such as vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and basic FGF; angiogenesis factors, and anti-angiogenesis factors.
  • VEGF vascular endothelial growth factor
  • KGF keratinocyte growth factor
  • basic FGF basic FGF
  • angiogenesis factors e.g., fibroblast growth factor
  • anti-angiogenesis factors e.g., trechalose (U.S. Patent Nos. 4,891,319; 5,290,765; 5,693,788).
  • the cells and tissues of the present invention may also be engineered to express gene products which provide a protective function in vivo, such as those which would protect the cells from an inflammatory response and protect against rejection by the host's immune system, such as HLA epitopes, major histo-compatibility epitopes, immuno-globulin and receptor epitopes. epitopes of cellular adhesion molecules, cytokines, chemokines.
  • cells and tissues may be protected against rejection by the host's immune system by mutating or deleting genes encoding proteins that identify the cells or tissues as non-self.
  • the cells and tissues of the present invention may be engineered to express gene products such as TGF- ⁇ , including TGF ⁇ -1 , TGF ⁇ -2, TGF ⁇ -3, TGF ⁇ -4 and TGF ⁇ -5, which regulate growth and differentiation and accelerate wound healing (Noda et al. 1989, Endocrin. 124: 2991-2995; Goey et al. 1989, J. Immunol. 143: 877- 880, Mutoe et al. 1987, Science 237: 1333-1335).
  • Cells and tissues of the present invention may be engineered to express mitogens, such as PDGF which increase the rate of cellularity and granulation in tissue formation (Kohler et al. 1974, Exp. Cell. Res. 87: 297-301).
  • mitogens such as PDGF which increase the rate of cellularity and granulation in tissue formation
  • the cells and tissues of the present invention may be genetically engineered to express nucleotide sequences or fragments thereof of the following genes:
  • Fibroblast growth factors and de ⁇ vatives thereof including FGF-2 and bFGF (U.S. Patent No. 5,026,839); FGF-1 and Cys-modified FGF (U.S. Patent No.
  • EPO Erythropoietin
  • preproinsulin U.S. Patent No. 4,431,740
  • insulin analogs U.S. Patent Nos. 4,701,440, 5,008,241 , 5,149,777, 5,164,366, 5,324,641, 5,618,913, 5,716,927)
  • superactive insulin analogs 4,992,417)
  • insulin precursor analogs U.S. Patent No. 5,324,641.
  • the gene products may be engineered to be expressed by the cells and tissues of the present invention.
  • the gene products may be engineered to be expressed constitutively or in a tissue-specific or stimuli-specific manner.
  • the nucleotide sequences encoding gene products may be operably linked to promoter elements which are constitutively active, tissue-specific or induced upon presence of a specific stimuli.
  • regulatory promoter elements include tetracycline responsive elements, nicotine responsive elements, insulin responsive element, glucose responsive elements, interferon responsive elements, glucocorticoid responsive elements estrogen progesterone responsive elements, retinoid acid responsive elements, viral transactivators, early or late promoter of SV40 adenovirus, the lac system, the tip system, the TAC system, the TRC system, the promoter for 3-phosphoglycerate and the promoters of acid phosphatase.
  • artificial response elements could be constructed, composed of multimers of transcription factor binding sites and hormone- response elements similar to the molecular architecture of naturally-occurring promoters and enhancers (e.g., see Hen, W & Clarke, J Cell (1986) 45(3): 461-70).
  • Such artificial composite regulatory regions could be designed to respond to any desirable signal and be expressed in particular cell-types depending on the promoter/enhancer binding sites selected.
  • transcriptional control regions that exhibit tissue specificity that may be utilized to regulate expression of a gene, include but are not limited to: elastase I gene control region which is active in pancreatic acinar
  • SUBST ⁇ UTE SHEET (RULE 26) cells Swift et al., 1984, cell 38:639—646; Omitz et al, 1986, Cold Spring Harbor Symp. Quant. Bid. 50:399-409; MacDonald, 1987, Hepatology 7:42S-51S); insulin gene control region which is active in pancreatic beta cells (Hanahan. 1985, Nature
  • liver 15 which is active in liver (Kelsey et al., 1987, Genes and Devel. 1 :161 — 171); beta — globin gene control region which is active in myeloid cells (Magram et al., 1985, Nature 315:338— 340; Kollias et al., 1986, Cell 46:89—94); myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703 — 712); myosin light chain — 2 gene control region which is active in
  • promoters can be inducible or constitutive, including but not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981 , Nature 290,
  • the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22, 787-797), the herpes thymidine kinase promoter (Wagner, et al, 1981, Proc. Natl. Acad. Sci. U.S.A. 78, 1441-1445), the regulatory sequences of the metallothionein gene (Brinster, et al., 1982, Nature 296, 39-42), etc.
  • genes of the invention may be introduced into the cell using recombinant DNA technology well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing the target gene coding sequences and 35 appropriate transcriptional and translational control signals.
  • DNA capable of encoding the target gene product sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques desc ⁇ bed in "Ohgonucleotide Synthesis", 1984, Gait, M.J. ed., IRL Press. Oxford, which is incorporated by reference herein in its entirety
  • a va ⁇ ety of host-expression vector systems may be utilized to express gene(s) (i e , the first gene(s), the target gene(s), and additional genes) coding sequences of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, exhibit the target gene product of the invention in situ.
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3
  • harbo ⁇ ng recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionem promoter) or from mammalian viruses (e g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
  • a number of viral-based expression systems may be utilized.
  • the gene coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e g , the late promoter and t ⁇ partite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by m vitro or in vivo recombination Insertion in a non-essential region of the viral genome (e g., region El or E3) will result in a recombinant virus that is viable and capable of expressing target gene product in infected hosts, (e.g., See Logan & Shenk, 1984, Proc. Natl.
  • Specific initiation signals may also be required for efficient translation of inserted target gene product coding sequences These signals include the ATG initiation codon and adjacent sequences. In cases where an entire gene, including its own initiation codon and adjacent sequences, is inserted into the appropnate expression vector, no additional translational control signals may be needed.
  • exogenous translational control signals including, perhaps, the ATG initiation codon
  • the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert
  • exogenous translational control signals and initiation codons can be of a va ⁇ ety of o ⁇ gins. both natural and synthetic.
  • the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:516-544). For long term, high-yield production of recombinant proteins, stable expression is prefened.
  • cell lines which stably express the first gene product may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • Cell lines can then be analyzed for optimal expression levels of the target gene product. This method may advantageously be used to engineer cell lines which express the target gene products. Such cell lines would be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the target gene product.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci.
  • dhfr which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci.
  • the cells and tissues may be engineered to express an endogenous gene under the control of a heterologous regulatory element, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination, e.g., see Chappell, U.S. Patent No. 5,272,071 ; PCT Publication No. WO91/06667, published May 16. 1991, which are
  • gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic enginee ⁇ ng methods.
  • regulatory sequences may be comp ⁇ sed of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences
  • sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting, including polyadenylation signals.
  • the targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g , inserting a new promoter or enhancer or both upstream of a gene.
  • the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element.
  • the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occur ⁇ ng elements.
  • the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome.
  • the identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a conect homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker.
  • Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacte ⁇ al xanthine-guanme phospho ⁇ bosyl-transferase (gpt) gene.
  • TK Herpes Simplex Virus thymidine kinase
  • gpt bacte ⁇ al xanthine-guanme phospho ⁇ bosyl-transferase
  • a va ⁇ ety of p ⁇ mary or secondary cells or cell strains may be used, including stromal and parenchymal cells.
  • the cells may be either fibroblasts, epithelial cells, keratinocytes, chondrocytes, hepatocytes. mesenchymal cells, chondro progenitor cells, or parenchymal cells of various tissues, including stem cells.
  • a number of cell types may be used in accordance with the present invention, including but not limited to cells isolated from skin, bone marrow, liver, pancreas, kidney, adrenal and neurological tissue to name a few.
  • Other cell types that may be used in accordance with the present invention are immune cells, macrophages/ monocytes.
  • secondary cell lines may be used as engineered responsive cells and tissues in accordance with the present invention, including, but not limited to hepatic cell lines, such as Chang liver cells, or other cell lines such as CHO, VERO, BHK, Hela, COS, MDCK, 293, 373, and W138 cell lines.
  • the engineered cells and tissues of the present invention may be obtained from an animal, preferably a mammal, and most preferably a human being.
  • the cells or tissues used for transplantation or implantation in vivo are obtained from the same species that will receive them.
  • the cells and tissues of the present invention may be autologous or non-autologous to the patient receiving them.
  • the cells and tissues are autologous to the patient receiving them.
  • Stromal cells such as these fibroblasts may be derived from organs such as skin, liver, pancreas, etc. which can be obtained by biopsy (where appropriate). Parenchymal cells may be obtained from cell suspensions prepared by disaggregating the desired tissue using standard techniques. Again, where the engineered cells and tissues are to be used for transplantation or implantation in vivo, it is preferable to obtain the stromal or parenchymal cells from the patient's own tissues. Although not all cells that may be used in accordance with the present invention grow in monolayers, such as fibroblasts, but rather grow in suspension, such as migratory immune cells. These cell types can be cultured using a pre-established three- dimensional cell culture system as described in U.S. Patent No.
  • the stromal support matrix comprises stromal cells, such as fibroblasts, actively growing on a three-dimensional matrix. Stromal cells may be also include other cells found in loose connective tissue such as endothelial cells, macrophages, monocytes. adipocytes, pericytes, reticular cells found in bone marrow, etc.
  • the stromal matrix provides the support, growth factors and regulatory factors necessary to sustain long-term a time proliferation of cells in culture. When grown in this three-dimensional system, the proliferating cells mature and segregate properly to form components of adult tissues analogous to counterparts found in vivo.
  • the cells of the present invention may be engineered to express one or more target gene sequences of interest, and may then be introduced into a patient in appropriate positions. Alternatively, cells that express an unimpaired target gene of
  • MHC major histocompatibility complex
  • the cells to be administered are non-autologous cells, they can be administered using well known techniques that prevent a host immune response against the introduced cells from developing.
  • the cells may be introduced in an
  • the genetically engineered cells and tissues of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. Expression of a gene(s) of the present invention can be readily detected, e.g., by quantifying protein and/or RNA.
  • kinase assays include but not limited to kinase assays, immunoassays to , detect and or visualize gene expression (e.g., Western blot, immunoprecipitation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect gene expression by detecting and or visualizing respectively mRNA encoding a gene (e.g., Northern assays, dot blots, in situ hybridization, etc), etc.
  • the activity of a target gene product e.g., Western assays, dot blots, in situ hybridization, etc.
  • tissue culture supernatant from the genetically engineered cells or tissues on different cells in culture.
  • Known effects of the target gene such as the activation of a signaling molecule or the induction of a gene product, can be assessed by standard methods known to those of skill in the art.
  • mice prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • any animal model system known in the art may be used prior to administration to humans.
  • the engineered cells can be used, with or without other cells or cell types which may or may not be genetically engineered: (a) to produce three-dimensional tissues in vitro (for implantation in vivo, or for use as extracorporeal devices or for use in vitro, as
  • the cells can be formulated as an injectable preparation of cells mixed with extracellular matrix proteins, collagen or synthetic crosslinkable polymers.
  • the genetically engineered cells are grown on a three-dimensional framework to produce three-dimensional cultures analogous to tissue counterparts in vivo using the techniques described in the U.S. Patent No.s. 4,721,096; 4,963,489; and 5,443,950 each of which is incorporated by reference herein
  • a living stromal tissue is produced in vitro composed of stromal cells, such as fibroblasts, actively growing on a three-dimensional framework.
  • stromal cells such as fibroblasts
  • gentically engineered may be mixed with other cells including other cells found in loose connective tissue such as endothelial cells,
  • the three-dimensional stromal cell culture provides the support, growth factors, and regulatory factors necessary to sustain long term active proliferation of cells in culture. When grown in this three-dimensional system, the proliferating cells mature and segregate to form components of adult tissues analogous to counterparts in
  • This system is particularly advantageous for producing tissues for use in human subjects, since human stromal cells can be used to create a "human tissue" - i.e., the cells and the extracellular matrix proteins generated in the three-dimensional stromal tissue will be of human origin and less likely rejected by patient-recipients of the engineered tissue. 5
  • This embodiment of the invention is based, in part, on the discovery that growth of stromal cells in three dimensions will sustain active proliferation of cells in culture for longer periods of time than will monolayer systems. This may be due, in part, to the increased surface area of the three-dimensional framework which results in a prolonged period of subconfluency and active proliferation of stromal cells.
  • stromal cells elaborate proteins (e.g., extracellular matrix proteins which form the connective tissue of the stromal tissue), growth factors and regulatory factors necessary to support the long term proliferation of both stromal cells (and parenchymal cells) inoculated onto the stromal tissue.
  • proteins e.g., extracellular matrix proteins which form the connective tissue of the stromal tissue
  • growth factors e.g., growth factors and regulatory factors necessary to support the long term proliferation of both stromal cells (and parenchymal cells) inoculated onto the stromal tissue.
  • the three-dimensionality of the framework allows for a spatial distribution which more closely approximates conditions in vivo, thus allowing for the formation of microenvironments conducive to cellular maturation and migration.
  • stromal cells such as human fibroblasts
  • a three- dimensional framework composed of any material and/or shape that: (a) allows cells to attach to it (or can be modified to allow cells to attach to it); and (b) allows cells to grow in more than one layer.
  • a number of different materials may be used to form the framework, including but not limited to: nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE; teflon), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures, collagen, cellulose, gelatin, dextran, etc. Any of these materials may be woven into a mesh, for example, to form the three-dimensional matrix. Certain materials, such as nylon, polystyrene, etc., are poor substrates for cellular attachment.
  • nylon matrices could be treated with 0.1 M acetic acid, and incubated in polylysine, FBS, and/or collagen to coat the nylon.
  • Polystyrene could be similarly treated using sulfuric acid.
  • biodegradable materials such as polyglycolic acid, catgut suture material, collagen (such as collagen sponge), or gelatin, for example.
  • nondegradable materials such as nylon, dacron, polystyrene, polyacrylates. polyvinyls, teflons, cotton, etc. may be prefened.
  • a convenient nylon mesh which could be used in accordance with the invention is Nitex. a nylon filtration mesh having an average pore size of 210 ⁇ m and an average nylon fiber diameter of 90 ⁇ m (#3 — 210/36, Tetko, Inc., N.Y.).
  • fibroblasts comprising fibroblasts. with or without other cells and elements desc ⁇ bed below, are inoculated onto the framework.
  • fibroblasts may be derived from organs, such as skin, liver, pancreas, etc. which can be obtained by biopsy (where approp ⁇ ate) or upon autopsy.
  • fibroblasts can be obtained in quantity rather conveniently from any appropriate cadaver organ.
  • fetal fibroblasts can be used to form a "gene ⁇ c" three-dimensional stromal mat ⁇ x that will support the growth of a variety of different cells and/or tissues.
  • a "specific" stromal mat ⁇ x may be prepared by inoculating the three-dimensional framework with fibroblasts derived from the same type of tissue to be cultured and/or from a particular individual who is later to receive the cells and/or tissues grown in culture in accordance with the three-dimensional system of the invention.
  • Fibroblasts may be readily isolated by disaggregating an approp ⁇ ate organ or tissue which is to serve as the source of the fibroblasts. This may be readily accomplished using techniques known to those skilled in the art.
  • the tissue or organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighbonng cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage.
  • Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination.
  • the suspension can be fractionated into subpopulations from which the fibroblasts and/or other stromal cells and/or elements can be obtained. This also may be accomplished using standard techniques for cell separation including but not limited to cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elut ⁇ ation (counterstreaming cent ⁇ fugation ). unit gravity separation, countercunent
  • fibroblasts may, for example, be earned out as follows: fresh tissue samples are thoroughly washed and minced in Hanks balanced salt solution (HBSS) in order to remove serum. The minced tissue is incubated from 1-12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin After such incubation, the dissociated cells are suspended, pelleted by cent ⁇ fugation and plated onto culture dishes. All fibroblasts will attach before other cells, therefore, appropriate stromal cells can be selectively isolated and grown.
  • HBSS Hanks balanced salt solution
  • the isolated fibroblasts can then be grown to confluency, lifted from the confluent culture and inoculated onto the three- dimensional framework (see, Naughton et al., 1987, J Med 18(3&4):219-250). Inoculation of the three-dimensional framework with a high concentration of stromal cells, e.g., approximately 10 6 to 5 x 10 7 cells/ml, will result in the establishment of the three-dimensional stromal support in shorter periods of time
  • stromal cells may be added to form the three- dimensional stromal mat ⁇ x required to support long term growth in culture.
  • other cells found in loose connective tissue may be inoculated onto the three- dimensional support along with fibroblasts.
  • Such cells include but are not limited to endothelial cells, pe ⁇ cytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, etc.
  • These stromal cells may readily be de ⁇ ved from appropnate organs such as skin, liver, etc., using methods known in the art such as those discussed above.
  • stromal cells which are specialized for the particular tissue to be cultured may be added to the fibroblast stroma.
  • stromal cells of hematopoietic tissue including but not limited to fibroblasts, endothelial cells, macrophages/ monocytes, adipocytes and reticular cells, could be used to form the three-dimensional subconfluent stroma for the long term culture of bone manow in vitro.
  • Hematopoietic stromal cells may be readily obtained from the "buffy coat" formed in bone manow suspensions by cent ⁇ fugation at low forces, e.g., 3000 x g.
  • Stromal cells of liver may include fibroblasts.
  • g al cells could be used as the stroma to support the proliferation of neurological cells and tissues
  • g al cells for this purpose can be obtained by trvpsinization or collagenase digestion of embryonic or adult brian (Ponten and Westermark, 1980, in Federof. S. Hertz, L., eds, "Advances in Cellular Neurobiology,” Vol.l, New York, Academic Press, pp. 209-227).
  • the cultured cells are to be used for transplantation or implantation in vivo it is preferable to obtain the stromal cells from the patient's own tissues.
  • the growth of cells in the presence of the three-dimensional stromal framework may be further enhanced by adding to the framework, or coating the framework with proteins (e.g., collagens. elastic fibers, reticular fibers) glycoproteins, glycosaminoglycans (e.g., heparin sulfate, chondroitin-4 — sulfate, chondroitin — 6- sulfate. dermatan sulfate, keratin sulfate. etc.), cellular matrix, and/or other materials.
  • proteins e.g., collagens. elastic fibers, reticular fibers
  • glycoproteins e.g., heparin sulfate, chondroitin-4 — sulfate, chondroitin — 6- sulfate. dermatan sulfate,
  • the three- dimensional culture After inoculation of the stromal cells, the three- dimensional culture should be incubated in an appropriate nutrient medium. Many commercially available media such as RPMI 1640, Fisher's, Iscove's, Mccoy's, and the like may be suitable for use. It is important that the three- dimensional stromal culture be suspended or floated in the medium during the incubation period in order to maximize proliferative activity. In addition, the culture should be "fed” periodically to remove the spent media, depopulate released cells, and add fresh media.
  • the stromal cells grow linearly along and envelop the three-dimensional framework before beginning to grow into the openings, and elaborate growth factors and regulatory factors necessary to support the long term proliferation of the cells inoculated or the framework, as well as other proteins, such as collagen and other extracellular matrix proteins that form the stromal matrix.
  • the resulting stromal tissue can be used in vivo or may be additionally inoculated on the framework, as well as other proteins, such as collagen and other extracellular matrix proteins that form the stromal matrix.
  • the resulting stromal tissue can be used in vivo or may be additionally inoculated with parenchymal cells to produce a tissue analogous to tissue counterparts in vivo.
  • the openings of the framework should be of an appropriate size to allow the stromal cells to stretch across the openings. Maintaining actively growing stromal cells which stretch across the framework enhances the production of growth factors which are elaborated by the stromal cells, and hence will support long term cultures. For example, if the openings are too small, the stromal cells may rapidly achieve confluence, exhibit contact inhibition and cease production of the appropriate factors necessary to support proliferation and maintain long term cultures. If the openings are too large, the stromal cells may be unable to stretch across the opening; this will also decrease stromal cell production of the appropriate factors necessary to support proliferation and maintain long term cultures.
  • openings ranging from about 150 ⁇ m to about 220 ⁇ m will work satisfactorily.
  • other sizes may work equally well.
  • any shape or structure that allow the stromal cells to stretch and continue to replicate and grow for lengthy time periods will work in accordance with the invention.
  • the framework should preferably contain collagen types III, IV and I in an approximate ratio of 6:3: 1 in the initial matrix.
  • collagen types I and III are preferably deposited in the initial framework.
  • the proportions of collagen types deposited can be manipulated or enhanced by selecting fibroblasts which elaborate the appropriate collagen type. This can be accomplished using monoclonal antibodies of an appropriate isotype or subclass that is capable of activating complement, and which define particular collagen types. These antibodies and complement can be used to negatively select the fibroblasts which express the desired collagen type.
  • the stromal cells used to inoculate the framework can be a mixture of cells which synthesize the appropriate collagen types desired.
  • the distribution and origins of the various types of collagen is shown in Table I.
  • the appropriate stromal cell(s) may be selected to inoculate the three-dimensional framework.
  • proliferating cells may be released from the framework. These released cells may stick to the walls of the culture vessel where they may continue to proliferate and form a confluent monolayer. This should be prevented or minimized, for example, by removal of the released cells during feeding, or by transferring the three-dimensional stromal framework to a new culture vessel. The presence of a confluent monolaver in the vessel will "shut down" the growth of cells in the three-dimensional culture. Removal of the confluent monolayer or transfer of the three-dimensional culture/ framework to fresh media in a new vessel will restore proliferative activity of the three-dimensional culture system. Such removal or transfers should be done in any culture vessel which has a stromal monolayer exceeding 25% confluency.
  • the culture system could be agitated to prevent the released cells from sticking, or instead of periodically feeding the cultures, the culture system could be set up so that fresh media continuously flows through the system.
  • the flow rate could be adjusted to both maximize proliferation within the three-dimensional culture, and to wash out and remove cells released from the matrix, so that they will not stick to the walls of the vessel and grow to confluence.
  • the released stromal cells can be collected and cryopreserved for future use.
  • the genetically engineered cells can be used to produce tissue equivalents using other systems known in the art, including but not limited to using synthetic scaffolding support systems (e.g., see U.S. Patent Nos.
  • bovine collagen matrices e.g. see U.S. Patent No. 4,485,096 incorporated herein by reference in its entirety
  • cross-linked collagen sponge matrices e.g., see PCT WO 91/16010, incorporated herein by reference in its entirety
  • genetically engineered cells are grown on extracellular matrix preparations composed of stromal cells which provide a three-dimensional cell culture system which may be formulated as an injectable, see PCT WO 96/39101, incorporated herein by reference in its entirety.
  • the cells and tissues of the present invention are grown using polymerizable or cross linking hydrogels as described in U.S. Patent Nos. 5.709.854; 5,516,532; 5,654,381 ; and WO98/52543. each of which is incorporated herein by reference in their entirety. Examples of materials which can be used to form a hydrogel include modified alginates.
  • Alginate is a carbohydrate polymer isolated from seaweed, which can be cross-linked to form a hydrogel by exposure to a divalent cation such as calcium, as described, for example in WO 94/25080. the disclosure of which is incorporated herein by reference. Alginate is ionically cross-linked in the presence of divalent cations, in water, at room temperature, to form a hydrogel mat ⁇ x
  • modified alginates refers to chemically modified alginates with modified hydrogel properties.
  • polvsaccha ⁇ des which gel by exposure to monovalent cations, including bactenal polysaccha ⁇ des. such as gellan gum. and plant polysaccha ⁇ des, such as canageenans, may be cross-linked to form a hydrogel using methods analogous to those available for the cross-linking of alginates desc ⁇ bed above
  • hyaluronic acids refers to natural and chemically modified hyaluronic acids
  • Modified hyaluronic acids may be designed and synthesized with preselected chemical modifications to adjust the rate and degree of cross-linking and biodegradation.
  • Covalently cross-linkable hydrogel precursors also are useful.
  • a water soluble polyamine such as chitosan
  • a water soluble dnsothiocyanate such as polyethylene glycol dnsothiocyanate
  • polymers may be utilized which include substituents which are cross-linked by a radical reaction upon contact with a radical initiator
  • polymers including ethylenically unsaturated groups which can be photochemically cross-linked which may be utilized, as disclosed in WO 93/17669, the disclosure of which is incorporated herein by reference.
  • water soluble macromers that include at least one water soluble region, a biodegradable region, and at least two free radical-polyme ⁇ zable regions, are provided Examples of these macromers are PEG-oligolactyl-acrylates, wherein the acrylate groups are polyme ⁇ zed using radical initiating systems, such as an eosin dye, or by b ⁇ ef exposure to ultraviolet or visible light
  • water soluble polymers which include cirmamoyl groups which may be photochemically cross-linked may be utilized, as disclosed in Matsuda et al , AS AID Trans , 38 154-157 (1992).
  • the prefened polymerizable groups are acrylates, diacrylates, ohgoacrylates, dimethacrylates, ohgomethacrylates, and other biologically acceptable photopolyme ⁇ zable groups
  • Acrylates are the most prefened active species polymenzable group
  • Naturally occur ⁇ ng and synthetic polymers may be modified using chemical reactions available in the art and desc ⁇ bed, for example, in March, "Advanced Organic Chemistry", 4 tn Edition. 1992. Wiley-Interscience Publication, New York.
  • Polyme ⁇ zation is preferably initiated using photoinitiators
  • Useful photoinitiators are those w hich can be used to initiate polyme ⁇ zation of the macromers without cytotoxicity and within a short time frame, minutes at most and most preferably seconds
  • Prefened dyes include erythrosin, phloxime. rose bengal, thonine, camphorquinone, ethyl eosin, eosin, methylene blue, ⁇ boflavin.
  • Suitable cocatalysts include amines such as N-methyl
  • the conditioned media or alternatively particular extracellular mat ⁇ x proteins elaborated into the media, are used to provide an excellent
  • the naturally secreted extracellular mat ⁇ x provides the conditioned media with type I and type III collagens, fibronectm, terascin, glycosaminologycans, acid and basic FGF. TGF- and TGF- ⁇ , KGF, ⁇ ersican, deco ⁇ n and va ⁇ ous other secreted human dermal mat ⁇ x proteins Similarly, the conditioned cell media of the invention or the extracellular mat ⁇ x proteins de ⁇ ved from the 0 conditioned media may be used to coat conventional implantation devices, including vascular prosthesis, in surgical approaches to conect defects in the body — resulting in supe ⁇ or implantation devices The implants should be made of biocompatible, inert materials, that replace or substitute for the defective function and made of either non-biodegradable mate ⁇ als or biodegradable mate ⁇ als By coating implantation 5 devices with the medium containing these extracellular proteins, the implant invites proper cellular attachments resulting in supe ⁇ or tissue at the implantation site Thus, sutures,
  • the cells or tissues are encapsulated as desc ⁇ bed, e g , in U S Patent Nos 5,874,099, 4,744.933, and 4,409.331. each of which is incorporated herein by reference in their entiretv
  • the encapsulation of geneticallv 5 engineered cell and tissues of the present ⁇ n ⁇ ention (l) protects cells or tissues from destruction bv the host's immune system, ( ⁇ ) enables nut ⁇ ents and oxygen to diffuse
  • the capsule in which the cells and tissues are encased is biodegradable. In another embodiment, the capsule in which the cells or tissues are encased provides the shape for the development of new organs, see e.g., U.S. Patent No. 5,770,417.
  • the engineered responsive cells and tissues of the present invention have a variety of applications including delivery of gene products locally or systematically.
  • the engineered responsive cells and tissues can be used for purposes of injection, transplantation or implantation in vivo.
  • Cells or tissues injected, transplanted or implanted in vivo can be autologous or non-autologous.
  • cells or tissues of the present invention can be primary or secondary cells or immortalized cell lines.
  • the cells/tissues and the stromal cells and elements are obtained from the individual who is to receive the transplant or implant. This approach might be especially advantageous where immunological rejection of the transplant and/or graft versus host disease is likely.
  • fibroblasts and other stromal cells and/or elements can be derived from the same type of tissue to be cultured in the three-dimensional system. This might be advantageous when culturing tissues in which specialized stromal cells may play particular structural/functional roles, e.g., glial cells of neurological tissue, Kupffer cells of liver etc.
  • Transfected primary or secondary cells or cell strains as described herein have wide applicability as a vehicle or delivery system for therapeutic products, such as enzymes, hormones, cytokines, antigens, antibodies, clotting factors, regulatory proteins, transcription proteins, receptors, structural proteins, novel (non-naturally occurring) proteins and nucleic acid products, such as anti-sense RNA, ribozymes, and engineered
  • therapeutic products such as enzymes, hormones, cytokines, antigens, antibodies, clotting factors, regulatory proteins, transcription proteins, receptors, structural proteins, novel (non-naturally occurring) proteins and nucleic acid products, such as anti-sense RNA, ribozymes, and engineered
  • transfected primary or secondary cells can be used to supply a therapeutic protein, including but not limited to, Factor VIII, Factor IX, erythropoietin, alpha- 1 antitrypsin, calcitonin, glucocerebrosidase, growth hormone, low density lipoprotein (LDL), apolipoprotein E. IL-2 receptor and its antagonists, insulin, globin. immunoglobulins. catalytic antibodies, the interleukins. insulin-like growth factors. superoxide dismutase. immune responder modifiers, parathyroid hormone and interferon, nerve growth factors, tissue plasminogen activators, and colony stimulating factors.
  • transfected primary and secondary cells can be used to immunize an individual (i.e., as a vaccine).
  • a secreted protein with predominantly systemic effects a secreted protein with predominantly local effects
  • a membrane protein imparting new or enhanced cellular responsiveness membrane protein facilitating removal of a toxic product
  • a membrane protein marking or targeting a cell an intracellular protein; an intracellular protein directly affecting gene expression; an j o intracellular protein with autolytic effects; gene product-engineered DNA which binds to or sequesters a regulatory protein; a ribozyme; and antisense-engineered RNA to inhibit gene expression.
  • the primary or secondary cells produced according to the present invention can be used to administer therapeutic proteins (e.g., hormones, enzymes, clotting factors) which are known in the art.
  • therapeutic proteins e.g., hormones, enzymes, clotting factors
  • J5 are presently administered intravenously, intra-muscularly or subcutaneously, which require patient cooperation and. often medical staff participation.
  • transfected primary or secondary cells of the present invention produce the therapeutic product as it would normally be produced.
  • transfected primary or secondary cells of the present 0 invention is that by controlling the number of cells introduced into an individual, one can control the amount of the product delivered to the body. In addition, in some cases, it is possible to remove the transfected cells or graft if there is no longer a need for the product.
  • a further advantage of treatment by use of transfected primary or secondary cells of the present invention is that production of the therapeutic product can be regulated such as 5 through the administration of zinc, steroids or an agent which affects translation or transcription of a protein, product or nucleic acid product or affects the stability of a nucleic acid product.
  • the cells or tissues are engineered to express a target gene product in response to a physiological change in glucose, insulin or glucagon levels.
  • cells and tissues are genetically engineered to express a first gene product that responds to a physiological change in glucose, insulin or glucagon levels, and in response to the physiological change induces
  • the cells of the present invention are engineered to respond to high levels of glucose and upon stimulation with glucose would express insulin.
  • the cells and tissues may be
  • GLP-1 derivatives include truncated derivatives GLP-1 (7-37), GLP-1 (7-36), GLP-1 (7-35) GLP-1 (7-34) and other truncated carboxy-terminal aminated derivatives and derivatives of GLP-1 which have amino acid substitutions, deletions, additions or other alterations (e.g., addition of a non-amino acid component) j o which result in biological activity or stability in the blood which is substantially the same as that of a truncated GLP-1 derivative or enhanced biological activity or stability in the blood (greater than that of a truncated GLP-1 derivative).
  • GLP-1 derivatives also known as insulinotropins or incretins, are normally secreted into the circulation by cells in the gastrointestinal tract.
  • these peptides function to j 5 stimulate insulin secretion and inhibit glucagon secretion from the endocrine pancreas, as well as increase insulin sensitivity in peripheral tissues (Goke, 1991 Eur. J. Clin. Inv. 21 :135-144; Gutniak et al., 1992 New Engl. J. Med. 326:1316-1822).
  • NIDDM non- insulin dependent diabetes mellitus
  • the cells and tissues are genetically engineered to be responsive to hypoxia.
  • the cells and tissues are engineered to express a receptor that responds to hypoxia and in turn induces the expression of VEGF and/or Flt-1 , which is under the control of a hypoxia-inducible enhancer element such as disclosed in, e.g. , Gerber et al, 1997, J. of Biological Chemistry
  • cells are engineered to respond to high levels of cholesterol circulating as low density lipoprotein particles or LDLs.
  • the cells may be engineered to express the LDL receptor, which would bind apolipoproteins B and
  • apolipoprotein E or apolipoprotein Al which function to bind cholesterol and direct it to the liver for degradation.
  • the cells and tissues are engineered to be responsive to a viral protein.
  • the cells or tissues may be engineered to express a cell surface receptor for the target virus, e.g. the CD4 or fusin receptor for HIV, and express an anti-HIV agent, e.g. a ribozyme targeted to HIV gag under the control of a HIV tat responsive promoter.
  • an anti-HBV agent e.g. a ribozyme targeted to HIV gag under the control of a HIV tat responsive promoter.
  • liver cells which express the receptor for Hepatitis B Virus (HBV) may be engineered to express an anti-HBV agent, e.g.
  • the therapeutic cells and tissues are engineered to be hyper-responsive to a viral protein.
  • the cells or tissues may be engineered to express a high-affinity cell surface receptor for the target virus, e.g. a modified form of CD4 or fusin receptor for HIV, and express an anti-HIV agent, e.g. a ribozyme targeted to HIV gag under the control of a HIV tat responsive promoter.
  • responsive cells and tissues are engineered to express an antigenic polypeptide and interleukin-2 (IL-2) in response to a viral infection.
  • a cell may be engineered to express the cell surface receptor for HTLV, HBV or HIV, and additionally be engineered to express the HTLV surface antigen, the HBV surface antigen or the HIV env protein and IL-2 under the control of the viral transactivator.
  • the cells and tissues secrete IL-2 which activates an immune response and viral antigens to prime the immune response.
  • therapeutic cells and tissues are engineered to secrete a gene product to replace a deleted or mutated gene product as a result of genetic or hereditary disease.
  • the cells and tissues would express Factor VIII; Hemophilia B, the cells and tissues would express Factor IX; Alpha thalessemia, the cells and tissues would express alpha globin; Beta thalassemia, the cells and tissues would express beta globin; Nieman- Pick disease, the cells and cells and tissues would express sphingomyelinase; Tay-Sachs disease, the cells and tissues would express lysosomal hexosaminidase.
  • cells are engineered to secrete a gene product to augment deficient or defective gene expression.
  • Apolipoprotein E isoform deficiencies have been associated with susceptibility to Alzheimer's disease. In this case
  • the engineered cells and tissues could be used to secrete the deficient ApoE isoform.
  • carbonic anhydrase II deficiency is associated with a va ⁇ ety of genetic and acquired kidney diseases.
  • the engineered cells and tissues could be used to secrete carbonic anhydrase II.
  • stromal cells such as fibroblasts
  • the stromal cells are transfected with nucleotides encoding the reporter gene under the control of viral regulatory elements, such as HIV tat.
  • the stromal cells are cotransfected with nucleotides encoding a cell surface receptor for the particular virus e.g., CD4 or fusin.
  • the transfected cells are grown in culture using a three-dimensional cell and
  • the bioreactor is grafted onto the subject.
  • Such a bioreactor would be useful in the case of a subject who had been exposed to the human immune deficiency virus (HIV) to determine when and if a viral infection would develop.
  • the subject would apply a formulation containing the reporter gene substrate to assay for the presence of the virus — once a positive result, as demonstrated by a change in the color of the skin graft, o was determined the subject would know to seek medical attention.
  • the cells and tissues are engineered to be responsive to a tumor antigen.
  • This embodiment of the invention would be useful for a subject that was in a high risk group for a particular type of cancer — or for a subject that had been previously treated for a cancer to determine if there was a recunence of the 5 cancer.
  • stromal cells may be engineered to express a cell surface receptor for the antigen, PMSA, which is associated with prostate cancer.
  • the cells are also be engineered to express a reporter gene under the control of an inducible promoter which would respond to the receptor binding PMSA.
  • These stromal cells are grown in three- dimensional culture systems to a size approp ⁇ ate for a skin patch.
  • Such a skin patch could o be grafted to a patient who had been treated for prostate cancer in order for the patient to self-monitor for recunence of the cancer.
  • the cells are engineered to respond to breast tumor antigens, liver tumor antigens, colon cancer antigens, lung cancer antigens etc.
  • the cells and tissues are genetically 5 engineered to be responsive to controlled substances such as cocaine and heroine.
  • the engineered cells are encapsulated in a skin patch which enables an individual (e.g., the police) to detect substance abuse.
  • the genetically engineered cells and tissues of the present invention are used to identify agents that modulate (e.g., upregulate or downregulate) the expression of a gene directly or indirectly.
  • agents that modulate e.g., upregulate or downregulate
  • the genetically engineered cells and tissues of the present invention are used to identify agents that modulate (e.g., upregulate or downregulate) the expression of a gene directly or indirectly.
  • JO engineered cells and tissues of the present invention can be used to identify agents that modulate the expression of a target gene.
  • agents, candidate compounds or test compounds include, but are not limited to, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules or other drugs.
  • modulators of the expression of a gene are j identified by contacting the engineered cells or tissues of the present invention with a candidate compound and determining the expression of the mRNA or protein encoded by the gene. The level of expression of the mRNA or protein in the presence of the candidate compound is compared to the level of expression of the selected mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as 0 a modulator of expression of the gene product based on this comparison. For example, when expression of the mRNA or protein encoded by a gene is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the mRNA or protein expression. Alternatively, when expression of the mRNA or protein encoded by the gene is less 5 (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the mRNA or protein expression.
  • the level of expression of an mRNA or protein encoded by a gene can be determined by methods known to those of skill in the art. For example, mRNA expression can be assessed by Northern blot analysis or RT-PCR. and protein levels can be assessed 0 by western blot analysis.
  • the genetically engineered cells and tissues of the present invention are used to identify agents that modulate the activity of a gene product.
  • the genetically engineered cells and tissues of the present invention are used to identify agents that modulate the activity of a target gene product.
  • modulators of the activity of a gene product are identified by contacting the engineered cells or tissues of the present invention with a test compound or a control compound (e.g., H 2 0) and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the gene product or a biologically active portion thereof. The activity of the gene product can be assessed by a test compound or a control compound (e.g., H 2 0) and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the gene product or a biologically active portion thereof. The activity of the gene product can be assessed by a test compound or a control compound (e.g., H 2 0) and determining the ability of the test compound to modulate (
  • detecting induction of a cellular second messenger of the gene product e.g., intracellular Ca2+. diacylglycerol, IP3, etc.
  • detecting catalytic/enzymatic activity of the target on an appropriate substrate detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for j o example, cellular differentiation, or cell proliferation.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • detecting a cellular response for j o example, cellular differentiation, or cell proliferation.
  • the candidate compound can then be identified as a modulator of the activity of a gene product by comparing the affects of the candidate compound to the control compound.
  • the genetically engineered cells and tissues of the present invention are used to identify pharmaceutical compositions that modulate the expression of a gene.
  • the genetically engineered cells and tissues of the present invention are used to identify pharmaceutical compositions that modulate the expression of a target gene.
  • the genetically 0 engineered cells and tissues of the present invention are implanted or injected in non- human animals (e.g., mice, rats, monkeys, rabbits, and guinea pigs), a pharmaceutical composition or a control composition is administered to the animals, and the effect of a pharmaceutical composition on gene expression is determined.
  • a pharmaceutical composition that alters the expression of a gene can be identified by comparing the level 5 of selected mRNA and/or protein expression (i.e., the mRNA or protein encoded by the gene) of an animal treated with a control composition to an animal treated with a pharmaceutical composition. Techniques know to those of skill in the art can be used to determine the mRNA and protein levels, for example, in situ hybridization.
  • the animals may or may not be sacrificed to assay the affects of a pharmaceutical composition.
  • the genetically engineered cells and tissues of the present invention are used to identify pharmaceutical compositions that modulate the activity of a gene product.
  • the genetically engineered cells and tissues of the present invention are used to identify pharmaceutical compositions that modulate the activity of a target gene product.
  • 25 genetically engineered cells and tissues of the present invention are implanted or injected in non-human animals (e.g., mice. rats, monkeys, rabbits, and guinea pigs), a pharmaceutical composition or a control composition is administered to the animals, and the effect of a pharmaceutical composition on the activity of a gene product is determined.
  • a pharmaceutical composition that alters the activity of a gene product can be identified by comparing the by animals treated with a control composition to an animal treated with a pharmaceutical composition.
  • the activity of the gene product can be assessed by detecting induction of a cellular second messenger of the gene product (e.g., intracellular Ca2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cellular differentiation, or cell proliferation.
  • a reporter gene e.g., a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • detecting a cellular response for example, cellular differentiation, or cell proliferation.
  • Three-dimensional, fibroblast cultures using cells transfected with Factor IX were used to assess the characteristics of the engineered fibroblasts as a gene delivery system.
  • Therapeutic fibroblasts were engineered by introducing the Factor IX nucleotide sequences via transfection with retroviral vectors carrying Factor IX driven by the long terminal repeat regulatory elements.
  • the transfected fibroblasts were grown into three-dimensional culture using both vicryl and nylon as scaffolds. These cultures secreted Factor IX into the medium at measurable levels for at least 12 days. At harvest the cultures were transplanted subcutaneously to nude mice and the blood concentrations recorded.
  • the engineered fibroblasts that secreted growth hormone was constructed using cells transfected with retroviral vectors carrying the rat growth hormone gene driven by the CMV promoter.
  • ulcers A major complication of ulcers is the high concentration of neutrophil elastase to be found in them that destroys growth factors and receptors (Grinnell, F. & M. Zhu, 1996
  • Tissue engineered products that inhibit neutriphil elatase can be devised by inducing the cells to secrete a neutrophil elastase inhibitor such as elafin (van Bergen. B.H. et al.. 1996 Arch. Dermatol. Res., 288(8): 458-62. or HEI
  • Genes for elafin or a related portease inhibitor might be transfected into a three-dimensional tissue construct on a suitable controllable or non-controlled promoter, and used to reduce tissue destruction in several diseases arising from abnormalities in tissue repair. Examples of diseases to be j 0 treated include ulcers, tumor metastasis, lung fibrosis etc.
  • the best mode of application of short-term gene expression is to transfect three- dimensional cultures using adenoviral vectors carrying genes optimized for human codon j 5 usage, driven by a powerful promoter, such as CM V, collagen, actin, RS V that may or may not shut down.
  • the cells are then grown in a three-dimensional scaffold to form a tissue.
  • the scaffold may be degradable, slowly degradable scaffolds must be removed, but make it easier to take out the transplanted tissue, as the cells do not move away as much. Degradable scaffolds will eventually cease to contain the transplant. However, they do not 0 need to be removed.
  • the rate of degradation of the scaffold can be adjusted to provide the optimal relationship between ease of removal, cell localization and convenience.
  • EXAMPLE METHODS FOR ALTERING DIFFERENTIATION The differentiation of cells depends on specific patterns of gene expression. These 5 patterns are in many cases induced through growth factor and cytokine responses. However, in every case, the surface reactions must be translated through asignal transduction pathway to the nucleus. Thus cellular differentiation might be controlled either through external proteins or through intracellular factors. Transfection provides a means to bring about the controlled expression of a secreted or intracellular protein. In 0 general, DNA molecules are easier to prepare and purify than proteins.
  • a culture might be transfected so as to cause the transient expression of a secreted growth factor that would then induce the appropriate response, or directly by inducing synthesis of an intracellular switching protein, such as a master gene.
  • an intracellular switching protein such as a master gene.
  • Slowly cycling cells are of interest because this population includes stem cells.
  • Slowly cycling cells might be enriched by transfecting cells with herpes simplex virus thymidine kinase. using a viral vector such as a retroviral vector that is unable to integrate in non-dividing cells. Cells in which the vector has been able to integrate are destroyed with gangcyclovir, thus enriching the population for stem cells. This technique might be combined with other stem cell selection methods.
  • telomere may have considerable disadvantages in therapeutic applications.
  • a way to avoid this is to insert the telomerase gene between loxP sites that can be activated to recombine, and thus excise the telomerase gene, by induction of ere.
  • this method can provide reliable reversible immortality on the cells. Such a technique could be applied to cells on a scaffold.
  • EXAMPLE METHODS FOR REGULATING THE EXPRESSION OF A TARGET GENE Stromal cells co-transfected with pCI-CD4 and pHIVtat-ribozyme are grown in a three-dimensional cell culture system.
  • the pCI-CD4 construct consists of the human CD4 cloned into the pCI mammalian expression vector (Promega Corporation, Madison, Wiscosin), and the pHIVtat-ribozyme construct consists of an HIV-ribozyme (see, e.g., Wong-Staal. F.
  • HIV-tat inducible expression vector see, e.g., Frasier. C. et al., 1998, Gene Ther 12: 1665-76.
  • the stromal cells are encased in a biocompatible capsule such as described in U.S. Patent No. 5,874.099 and implanted in vivo. HIV binds to the constitutively expressed CD4 molecule on the encapsulated stromal cells, infects the cells, and the expression of the HIV tat protein induces the expression of the HIV-ribozyme, which cleaves HIV RNA.
  • the advantage of this system is that HIV-ribozymes will produced in response to the presence of the virus.
  • stromal cells can be co-transfected with pCI-CD4 andpHIVtat-GRP, grown in a three-dimensional cell culture system, and encapsulated.
  • the encapsulated cells are implanted / ' /; vivo in a position such that capsule is readily accessible and retrievable.
  • the expression of the CD4 receptor allows the stromal cells to become infected with HIV, and the expression of HIV tat protein induces the expression of green fluorescent protein ("GFP”), which is by detectable fluorescence microscopy.
  • GFP green fluorescent protein
  • This system j o enables physicians to monitor individuals that have been exposed to HIV in order to determine infection, and enables physicians to determine the best therapeutic treatment regimen.
  • Stromal cells are co-transfected with pCI-TR and pTRE-GRP by techniques known to those of skill in the art.
  • the pCI-TR construct consists of the nucleotide sequence encoding human thyroid hormone receptor ("TR") cloned into the pCI mammalian expression vector (Promega Corporation, Madison, Wiscosin), and the pTRE-lacZ construct consists of the nucleotide sequence encoding lacZ cloned into an expression vector. LacZ expression is under the control of the thyroid hormone response element, which has been engineered to be sensitive to high concentrations of thyroid hormone.
  • TR human thyroid hormone receptor
  • Stromal cells expressing the genes are selected for and the stably transfected cells are encapsulated in a biocompatible patch that adheres to the skin.
  • the constitutive expression of the thyroid receptor enables the cells to respond to thyroid hormones (e.g., T4 and T3) 5 and, in response, induce the expression of lacZ.
  • the expression of lacZ is detectable by applying X-gal or something analogous and assessing the color of the patch. If the patch turns blue, then the thyroid hormone concentrations are high and medical attention is required.
  • This system enables the recipient or his/her physician to monitor the thyroid hormone concentrations in individuals with thyroid diseases and/or disorders including, 0 but not limited to, hyperthyroidism (e.g., Grave's disease) and hypothyroidism conditions

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Abstract

La présente invention concerne des cellules génétiquement modifies capables d'exprimer un gène cible en réponse à un 'signal environnemental' ou à un 'stimulus', ainsi que leur utilisation pour la production de tissus tridimensionnels ou de préparations injectables pour la réparation, le remplacement ou l'amélioration de tissus, et pour l'administration de thérapies géniques in vivo. En particulier, cette invention concerne des bioréacteurs cellulaires et tissulaires conçus pour exprimer un produit génique cible qui renseigne sur un état physiologique déterminé, accentue l'expression déficiente ou défectueuse du produit génique ou déploie une activité anti-virale, anti-bactérienne, anti-microbienne ou anti-cancéreuse en réponse à un 'signal environnemental' ou à un 'stimulus' donné.
PCT/US1999/016961 1998-07-28 1999-07-28 Cellules genetiquement modifiees et tissus WO2000006204A2 (fr)

Priority Applications (5)

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KR1020017001189A KR20010085313A (ko) 1998-07-28 1999-07-28 유전자조작된 세포와 조직
EP99938808A EP1100548A2 (fr) 1998-07-28 1999-07-28 Cellules genetiquement modifiees et tissus
AU53212/99A AU5321299A (en) 1998-07-28 1999-07-28 Genetically engineered cells and tissues
JP2000562057A JP2002521046A (ja) 1998-07-28 1999-07-28 遺伝子工学的に操作された細胞および組織
CA002338487A CA2338487A1 (fr) 1998-07-28 1999-07-28 Cellules genetiquement modifiees et tissus

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WO2001068828A2 (fr) * 2000-03-13 2001-09-20 Engene, Inc. Compositions et methodes destinees a l'expression regulee d'une proteine dans l'intestin
JP2003533984A (ja) * 2000-04-17 2003-11-18 エーバーハルト−カルルス−ウニヴェルズィテート テュービンゲン ウニヴェルズィテートクリーニクム 可逆的不死化
EP2383334A1 (fr) * 2009-01-23 2011-11-02 Osaka University Cellule nourricière destinée à induire des cellules cibles
WO2020112904A1 (fr) * 2018-11-26 2020-06-04 Massachusetts Institute Of Technology Compositions et procédés pour induire une tolérance immunitaire

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AU2007285472B2 (en) 2006-03-30 2013-10-24 Engene, Inc. Non-viral compositions and methods for transfecting gut cells in vivo
JP6206792B2 (ja) * 2013-04-03 2017-10-04 誠一 横尾 培地及び細胞の培養方法
CN114724643A (zh) * 2021-01-06 2022-07-08 腾讯科技（深圳）有限公司 一种多肽化合物的筛选方法以及相关装置

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WO1996040175A1 (fr) * 1995-06-07 1996-12-19 Advanced Tissue Sciences, Inc. Systeme de culture tridimensionnel base sur les cellules du stroma et destine a la formation de tubes, de tendons, de ligaments et de structures correctives
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WO2001068828A2 (fr) * 2000-03-13 2001-09-20 Engene, Inc. Compositions et methodes destinees a l'expression regulee d'une proteine dans l'intestin
WO2001068828A3 (fr) * 2000-03-13 2002-06-13 Engene Inc Compositions et methodes destinees a l'expression regulee d'une proteine dans l'intestin
JP2004500104A (ja) * 2000-03-13 2004-01-08 エンジーン，インコーポレーテッド 腸管における調節されたタンパク質発現のための組成物と方法
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EP2298357A1 (fr) * 2000-03-13 2011-03-23 Engene, Inc. Compositions et methodes destinées a l'expression regulée d'une proteine dans l'intestin
JP2003533984A (ja) * 2000-04-17 2003-11-18 エーバーハルト−カルルス−ウニヴェルズィテート テュービンゲン ウニヴェルズィテートクリーニクム 可逆的不死化
EP2383334A1 (fr) * 2009-01-23 2011-11-02 Osaka University Cellule nourricière destinée à induire des cellules cibles
EP2383334A4 (fr) * 2009-01-23 2013-01-16 Univ Osaka Cellule nourricière destinée à induire des cellules cibles
WO2020112904A1 (fr) * 2018-11-26 2020-06-04 Massachusetts Institute Of Technology Compositions et procédés pour induire une tolérance immunitaire

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CA2338487A1 (fr) 2000-02-10
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AU5321299A (en) 2000-02-21
EP1100548A2 (fr) 2001-05-23
KR20010085313A (ko) 2001-09-07

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