EP1893750A1 - Cellules amniotiques et leurs méthodes d'utilisation - Google Patents

Cellules amniotiques et leurs méthodes d'utilisation

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Publication number
EP1893750A1
EP1893750A1 EP06753014A EP06753014A EP1893750A1 EP 1893750 A1 EP1893750 A1 EP 1893750A1 EP 06753014 A EP06753014 A EP 06753014A EP 06753014 A EP06753014 A EP 06753014A EP 1893750 A1 EP1893750 A1 EP 1893750A1
Authority
EP
European Patent Office
Prior art keywords
nervous system
cells
hac
central nervous
human amniotic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP06753014A
Other languages
German (de)
English (en)
Other versions
EP1893750A4 (fr
Inventor
Lihe Guo
Tianjin Liu
Jiacai Wu
Xuesong Wang
Zhihua Jiang
Qin Huang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cell Star Bio-Technologies Co Ltd
Original Assignee
Cell Star Bio-Technologies Co Ltd
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Filing date
Publication date
Application filed by Cell Star Bio-Technologies Co Ltd filed Critical Cell Star Bio-Technologies Co Ltd
Publication of EP1893750A1 publication Critical patent/EP1893750A1/fr
Publication of EP1893750A4 publication Critical patent/EP1893750A4/fr
Withdrawn legal-status Critical Current

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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • 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
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C12N2830/006—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible

Definitions

  • the present invention relates to the fields of molecular biology, gene therapy, immunology and virology. More particularly, the invention relates to human amniotic cells comprising lentiviral vectors with an exogenous gene element, methods for making such cells and methods for treating diseases.
  • central nervous system diseases as cerebral ischemia, cerebral vascular disease, central nervous system injuries, hereditary diseases of the nervous system, degenerative diseases of the cerebral nervous system (for example, Parkinson's disease, Huntington's disease and Alzheimer's disease) and tumors of the cerebral nervous system.
  • central nervous system diseases for example, cerebral ischemia, cerebral vascular disease, central nervous system injuries, hereditary diseases of the nervous system, degenerative diseases of the cerebral nervous system (for example, Parkinson's disease, Huntington's disease and Alzheimer's disease) and tumors of the cerebral nervous system.
  • central nervous system diseases for example, central nervous system diseases that are a central nervous system.
  • central nervous system diseases for example, every year 16,400,000 people suffer from brain strokes, brain injuries and spinal cord injuries throughout the world.
  • 4,100,000 of these people die each year due to such maladies.
  • gene therapy has developed as a potentially potent method for treating many neurological diseases previously considered refractory to conventional approaches.
  • central nervous system diseases there are three strategies in gene therapy for treating central nervous system diseases.
  • One strategy is to decrease the activity of mutation recessive gene proteins with antisense technic and RNAi technic.
  • mutation recessive genes of central nervous system diseases express proteins and gene therapy can be used to down-regulate expression of theses proteins via antisense or RNAi to the mutated mRNA.
  • Another strategy is to compensate for loss- function enzymes or proteins of the brain by transferring genes.
  • neurons can be protected by delivery of proteins such as growth factors, antioxidants, HSP or anti-apoptotic molecules. Natsume et al., Exp.
  • virus vectors Five types are currently used in gene therapy: (1) retrovirus vectors, which only infect cells that can proliferate; (2) adenoviral (Ad) vectors that do not insert into the chromosomes of host cells and cannot express stably (Ad vectors can also produce immunogenicity); (3) adeno-associated viral (AAV) vectors, which cannot transport large exogenous fragments (less than 5 kb) or have high titers; (4) herpes simplex viruses (HSV) that only infect neuronal cells; and (5) lentiviral vectors.
  • retrovirus vectors which only infect cells that can proliferate
  • Ad adenoviral vectors that do not insert into the chromosomes of host cells and cannot express stably (Ad vectors can also produce immunogenicity)
  • Ad vectors can also produce immunogenicity
  • Ad vectors adeno-associated viral vectors, which cannot transport large exogenous fragments (less than 5 kb) or have high titers
  • the present invention in one aspect, relates to a population of human amniotic cells (HAC) comprising lentiviral vectors with at least one exogenous gene element.
  • HAC human amniotic cells
  • the exogenous gene elements of the vector are capable of being expressed by the cells.
  • the invention features a composition comprising these cells and at least one pharmaceutically acceptable carrier.
  • Another object of the present invention is to provide a method for transducing human amniotic cells.
  • Another object of the invention is to provide a method for treating central nervous system diseases using the human amniotic cells comprising lentiviral vectors with at least one exogenous gene element.
  • a method of the invention comprises administering human amniotic cells comprising lentiviral vectors with at least one exogenous gene element to a patient.
  • the invention also provides transfected human amniotic cells, which can be useful for gene therapy.
  • the exogenous gene element can be highly expressed in the human amniotic cells comprising lentiviral vectors.
  • These cells can effectively treat central nervous system diseases such as, for example, cerebral ischemia, cerebral vascular disease, central nervous system injuries, hereditary diseases of the nervous system, degenerative diseases of the cerebral nervous system (for example, Parkinson's disease, Huntington's disease and Alzheimer's disease), tumors of the cerebral nervous system and combinations thereof.
  • the cells can also be used to treat any of the diseases disclosed herein.
  • human amniotic cells according to the invention can minimize or eliminate rejection reactions or topical inflammations.
  • the human amniotic cells can be derived from, without limitation, a donor, tissue-cultures or subject (for example, patient).
  • lentiviral vector transduced HAC may also be used in the treatment or prophylaxis of diseases that include, without limitation, cerebral ischemia, cerebral vascular disease, central nervous system injuries, hereditary diseases of the nervous system, degenerative diseases of the cerebral nervous system (Parkinson's disease, Huntington's disease and Alzheimer's disease), tumors of the cerebral nervous system and combinations thereof.
  • the lentiviral vector transduced HAC of the invention can also be used in the treatment or prophylaxis of any disease state or malady disclosed herein.
  • prophylactic or “prophylaxis” relates to a reduction in the likelihood of the patient developing a disorder such as AD or proceeding to a diagnosis state for the disorder.
  • the lentiviral vector transduced HAC of the invention can be used prophylacticly as a measure designed to preserve health and prevent the spread or maturation of disease in a patient.
  • the invention also provides for methods of administering lentiviral vector transduced HAC to a patient in an effective amount for the treatment or prophylaxis of a disease such as, for example, CNS diseases.
  • the lentiviral vector transduced HAC of the invention can also be administered to a patient along with other conventional therapeutic measures or agents that maybe useful in the treatment or prophylaxis of, for example, CNS diseases.
  • a method for administering an effective amount of lentiviral vector transduced HAC of the invention to a patient suffering from or believed to be at risk of suffering from a disease.
  • the method also comprises administering, either sequentially or in combination with lentiviral vector transduced HAC of the invention, a conventional therapeutic agent in an amount that can potentially be effective for the treatment or prophylaxis of a CNS disease.
  • lentiviral vector transduced HAC of the invention can be administered to a patient in an amount or dosage suitable for treating a CNS disease.
  • a dosage or composition comprising lentiviral vector transduced HAC of the invention will vary depending on subject considerations. Such considerations include, for example, age, condition, sex, extent of disease, contraindications and concomitant therapies. An exemplary dosage or composition based on these considerations can also be adjusted or modified by a person of ordinary skill in the art.
  • Administration of lentivirai vector transduced HAC of the invention to a subject maybe local or systemic and accomplished intravenously, intraarterially, intrathecally (via the spinal fluid) or the like.
  • a “subject” is a mammal such as, for example, a human, and, preferably, a human suspected of having one or more CNS diseases.
  • the terms “subject” and “patient” are used interchangeably herein.
  • the lentivirai vector transduced HAC of the invention can also be administered in the form of a composition such as an injectable composition, but may also be formulated into well known drug delivery systems such as, for example, topical, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), intracisternal, intravaginal, intraperitoneal, local (powders, ointments or drops) or as a graft, buccal or nasal spray.
  • administration of lentivirai vector transduced HAC or compositions thereof may be local or systemic and accomplished intravenously, intraarterially, intrathecally (via the spinal fluid) or via a graft.
  • the administration of lentivirai vector transduced HAC to a subj ect can be by a general or local administration route.
  • the lentivirai vector transduced HAC or compositions thereof may be administered to the patient such that it is delivered throughout the body.
  • the lentivirai vector transduced HAC or compositions thereof can be administered to a specific organ or tissue of interest.
  • a typical composition for administration can comprise a pharmaceutically acceptable carrier for one or more lentivirai vector transduced HAC of the invention.
  • a pharmaceutically acceptable carrier includes such carriers as, for example, aqueous solutions and non-toxic excipients including salts, preservatives and buffers.
  • exemplary pharmaceutically acceptable carriers for lentivirai vector transduced HAC of the invention can also include non-aqueous solvents such as propylene glycol, polyethylene glycol, methoxypolyethylene glycol and vegetable oil or injectable organic esters such as ethyl oleate.
  • lentivirai vector transduced HAC can be conjugated to at least one pharmaceutically acceptable carrier.
  • An aqueous carrier can include, without limitation, water, alcoholic/aqueous solutions, saline solutions and parenteral vehicles such as sodium chloride or Ringer's dextrose.
  • Intravenous carriers for administration of lentiviral vector transduced HAC of the invention can include, for example, fluid and nutrient replenishers.
  • the pH and exact concentration of the various components for a composition comprising lentiviral vector transduced HAC can also be adjusted according to routine techniques known to those of ordinary skill in the art. Goodman and Gilman's The Pharmacological Basis for Therapeutics, 7th Edition.
  • the invention provides a kit comprising lentiviral vector transduced HAC.
  • the invention also provides a method for the treatment or prophylaxis of a CNS disease comprising administering to a patient in need thereof an effective amount of lentiviral vector transduced HAC.
  • the method can include providing a patient suffering from or believed to be at risk of suffering from a CNS disease.
  • the method may also comprise administering to the patient an effective amount of lentiviral vector transduced HAC of the invention.
  • the lentiviral vector transduced HAC of the invention can also be administered as part of a composition comprising a pharmaceutically acceptable carrier.
  • Effective amount refers to the amount required to produce a desired effect.
  • One example of an effective amount includes amounts or dosages that can be used to alleviate or minimize the effects of a CNS disease.
  • Another example of an effective amount includes amounts or dosages that yield acceptable toxicity and bioavailability levels for therapeutic use including, without limitation, the treatment or prophylaxis of a CNS disease.
  • lentiviral vector transduced HAC of the invention or compositions thereof may contain an additive such as pH controlling agents (for example, acids, bases, buffers), stabilizers (for example, ascorbic acid) or isotonizing agents (for example, sodium chloride).
  • pH controlling agents for example, acids, bases, buffers
  • stabilizers for example, ascorbic acid
  • isotonizing agents for example, sodium chloride
  • lentiviral vector transduced HAC can be used for ex vivo or in vivo gene therapy.
  • retrovirus, AAV, lentiviral, pseudotyped lentiviral or Ad vectors for the transduction of HAC can be administered to a subject in an effective amount.
  • vectors can be administered in a composition comprising a pharmaceutically acceptable carrier.
  • these vectors can be administered topically, orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops) or as a graft, buccal or nasal spray.
  • administration can also be local or systemic and accomplished intravenously, intraarterially, intrathecally (via the spinal fluid) or via a graft.
  • an enhancer sequence may be used to increase the level of expression.
  • exemplary growth factors encoded by exogenous gene elements include nerve growth factor, neurotrophic factors, brain derived growth factor (BDNF), neurotrophin (NT)-3, NT-4 and ciliary neuronal trophic factor (CNTF).
  • BDNF brain derived growth factor
  • NT neurotrophin
  • CNTF ciliary neuronal trophic factor
  • the invention also contemplates analogs, homologs, derivatives and variants of nerve growth factor, brain-derived neurotrophic factor, hypoxanthine guanine phosphoribosyltransferase, glia-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, bcl-2 or tetrahydrobiopterin synthase.
  • the invention contemplates analogs, homologs, derivatives and variants of exogenous gene elements encoding, for example, nerve growth factor, brain-derived neurotrophic factor, hypoxanthine guanine phosphoribosyltransferase, glia-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, bcl-2 or tetrahydrobiopterin synthase.
  • nerve growth factor for example, nerve growth factor, brain-derived neurotrophic factor, hypoxanthine guanine phosphoribosyltransferase, glia-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, bcl-2 or tetrahydrobiopterin synthase.
  • Exogenous elements can also be used to encode tyrosine hydroxylase, GTP-cyclohydrolase I, aromatic amino acid dopa decarboxylase, vesicular monoamine transporter 2 (VMAT2) or any suitable protein or antibody.
  • VMAT2 vesicular monoamine transporter 2
  • the invention contemplates transduction of HAC via, without limitation, one or more retrovirus, AAV, lentiviral, pseudotyped lentiviral, Ad vectors and combinations thereof.
  • retrovirus AAV
  • lentiviral pseudotyped lentiviral
  • Ad vectors Ad vectors and combinations thereof.
  • these vectors can be used either sequentially or in combination to transduce HAC.
  • a vector can also comprise one or more exogenous elements.
  • a population of human amniotic cells comprising one or more lentiviral vectors is provided.
  • the lentiviral vectors can comprise at least one exogenous gene element, which is expressed by the cells.
  • Exemplary exogenous gene elements can encode one of nerve growth factor, brain-derived neurotrophic factor, hypoxanthine-guanine phosphoribosyltransferase, glia-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L ⁇ amino acid decarboxylase, bcl-2 or tetrahydrobiopterin synthase.
  • the lentiviral vectors can comprise at least one controlling transcription fragment of an RNAi-inducible, Cre-loxP or doxycycline-inducible system.
  • the invention also provides a composition comprising one or more lentiviral vectors that include at least one exogenous gene.
  • a method of transducing a population of human amniotic cells with one or more lentiviral vectors comprises culturing the human amniotic cells.
  • an amnion can be separated from a placenta obtained from a donor.
  • the placenta can be extensively scraped out to remove the underlying tissues.
  • the amnion can optionally be treated with at least one enzyme.
  • the isolated cells can be cultured in a cell culture medium.
  • the method also comprises incubating at least one vector and the population of human amniotic cells.
  • a method for treating central nervous system diseases in a subject involves the use of a population of human amniotic cells comprising a lentiviral vector with an exogenous gene element.
  • CNS diseases include cerebral ischemia, cerebral hemorrhage, central nervous system trauma, hereditary diseases of the nervous system, degenerative diseases of the central nervous system (Parkinson's disease, Huntington's disease and Alzheimer's disease), neurodegenerative diseases, spinal cord trauma and neoplasms of the central nervous system.
  • Transduction or transfection is generally used to refer to the introduction of genetic material into a cell such as a human amniotic cell by using a vector, for example, a retrovirus, AAV, lentiviral, pseudotyped lentiviral or Ad vector.
  • a vector for example, a retrovirus, AAV, lentiviral, pseudotyped lentiviral or Ad vector.
  • vectors can be used for the transduction or transfection of HAC. These vectors include plasmid or viral vectors.
  • Retroviral vectors such as those based on Moloney murine leukemia virus (MoMLV) can be used.
  • other murine retroviral vectors that can be used include those based on murine embryonic stem cell virus (MESV) and murine stem cell virus (MSCV).
  • Lentiviral vectors a subclass of the retroviral vectors, can also be used for high- efficiency transduction and are able to transduce non-dividing cells, increasing the likelihood that the cells can be pluripotent.
  • Other groups of retroviruses such as spumaviruses are also capable of efficiently transducing non-dividing cells.
  • Additional types of viral vectors that can be used in the invention include adenoviral vectors, adeno- associated viral (AAV) vectors, SV40 based vectors or forms of hybrid vectors.
  • a wide variety of expression vectors are available for transferring gene elements, for example, endogenous or exogenous gene elements, encoding bioactive materials into HAC.
  • These expression vectors can be viral vectors such as modified or recombinant retroviruses, adenoviruses, lentiviruses, pseudotyped lentiviruses and adeno-associated viruses.
  • the expression vectors can be transfected to the cells via non-viral routes such as, without limitation, physical methods including electroporation, ultrasound, chemical, liposome-mediated, activated-dendrimer-mediated and calcium-phosphate techniques.
  • promoters useful in the invention include constitutive and inducible promoters.
  • such promoters can comprise CMV, SV40, retroviral LTR, EF, tetracycline inducible, inflammation induced, TNF- ⁇ and DLr-I promoters.
  • the invention may comprise various other components and means to facilitate the delivery of cells to a subject.
  • HAC can be provided within a medium in which the cells are preserved or maintained. Examples of such a medium include PBS, DMEM and any suitable type of cell culture medium.
  • the invention contemplates buffers such as, without limitation, HEPES, PBS or citrate-based buffers.
  • the invention can include dyes, packagings, kits, instructions for using transduced HAC, DMSO and glycerols for cryopreservation.
  • the transduced HAC of the invention can be used for, without limitation, therapy, diagnosis, cosmetics or any other applications.
  • exogenous elements can include, without limitation, growth factors, anti-microbial proteins, anti-inflammatory protein or protease inhibitors. Examples of growth factors can include PDGF, FGF-2, EGF, KGF-2, GM-C SF, TGF-b, IGF-I and HGH.
  • examples of anti-microbial proteins can include bactericidal/permeability- increasing proteins, defensin, collectin, granulysin, protegrin-1, SMAP-29, lactoferrin and calgranulin C.
  • examples of anti-inflammatory proteins can include interleul ⁇ n-1 receptor antagonists, interleukin-10, soluble TNF receptors and soluble CTLA4.
  • Protease inhibitors can also include, for example, TIMP-I, -2, -3, -A, PAI-I, PAI-2 and ecotin.
  • each of the vectors for transfection of HAC can comprise at least one promoter.
  • the exogenous gene elements of a vector can also comprise a marker sequence.
  • cells that can be transduced with a vector include those derived from or comprising, without limitation, mammalian tissues, epithelial cells, alveolar cells, bone marrow, cardiac muscles, connective tissues, ependymal cells, epithelial tissues, epithelial cells, epidermis, esophagus, fibroblasts, glial cells, hepatic cells, keratinocytes, leukocytes, lymphocytes, macrophages, mammary glands, melanocytes, monocytes, myoblasts, neurons, osteoblasts, osteogenic cells, pituicytes, plasma cells, skeletal muscles, smooth muscles, synoviocytes, umbilical tissues, HAC or combinations thereof.
  • the exogenous gene elements of a vector can also comprise a sequence encoding for a detectable marker
  • lentiviral vector transduced HAC can be used to prepare a graft composition.
  • a graft composition can include cells transfected by a vector comprising endogenous or exogenous gene elements.
  • a graft composition can also comprise a biocompatible matrix.
  • the HAC of a graft composition can be taken from a subject and transfected in vitro prior to reintroduction to the subject.
  • Exemplary biocompatible matrices for a graft composition include those that are natural or synthetic.
  • a graft composition can also comprise functional elements that can be either constitutive or inducible via physical or chemical stimuli to effect up or down regulation during therapy.
  • a graft composition can be administered, without limitation, via the gastrointestinal tract (orally or as a suppository), parentexally (intramuscular, intravenous or subcutaneous) or topically.
  • composition comprising vector transduced HAC and additional components.
  • components can include cells, genetically modified cells, proteins, peptides, non-protein bioorganic substances, therapeutic agents (antiinflammatories, antibiotics, antivirals, antineoplastics or antimycotics) and combinations thereof,
  • the vector transduced HAC of the invention can comprise at least one exogenous gene element that encodes for glia or brain-derived neurotrophic factors, which can be used in the prophylaxis or treatment of CNS diseases.
  • a population of transduced HAC or compositions thereof can be administered to a patient, for example, locally, at exemplary dosage levels in the range of about 10 3 cells to about 10 10 cells per day.
  • the specific dosage used can vary or may be adjusted as considered appropriate by a person of ordinary skill in the art. Without limitation, the dosage can depend on a number of factors including method of administration, requirements of the patient and severity of the disease being treated. The determination of optimum dosages for a particular patient is also well known to those of ordinary skill in the art.
  • Fig. 1 a map of lentiviral vectors used herein.
  • Vectors contain the post- transcriptional regulatory element of woodchuck hepatitis virus (WPRE or WHV) and central polypurine tract (cPPT), which enhance transgene expression as well as cis-acting elements, improving the efficiency of gene transfer.
  • AU vectors contain promoters: elongation factor 1-alfa promoter (EFl- ⁇ ) or human cytomegalovirus (CMV), which are robust transcriptional elements in most cell types.
  • EFl- ⁇ elongation factor 1-alfa promoter
  • CMV human cytomegalovirus
  • Fig. 2 efficient transfer, integration and sustained long-term expression of EGFP in HAC.
  • EGFP was evaluated on photographs that were taken with a fluorescence microscope (x 10) and transduction efficiency was measured by FACS. The EGFP expression was sustained for 5 weeks without obvious change, (Fig. 2B). The EGFP was integrated into genomic DNA and transcripted into mRNA that was detected by PCR analysis, (Fig. 2C). The same percent of GO-Gl cells before and after infection with vectors were shown, (Fig. 2D).
  • FIG. 3 lentiviral vector-mediated siGFP suppression in HAC. Photographs were taken with a fluorescence microscope (x 10) at 7 days after transfection of HAC with EGFP- expressing lentiviral vectors, (Fig. 3A), and efficiency was measured by FACS, (Fig. 3B). At 7 days after transfection of HAC with EGFP-expressing lentiviral vectors together with lentiviral vectors expressing siGFP, (Fig. 3C), absence of EGFP expression can be seen only in the group of siGFP-expressing vectors. The suppression of EGFP transcription was detected by RT-PCR analysis, (Fig. 3D).
  • Fig. 4 the EGFP was efficiently regulated by Cre-loxP system base on lentiviral vectors in HAC. EGFP was observed by fluorescence microscopy (x 10) after being transduced with lenti-EGFP, (Fig. 4A). Fluorescence was silenced by cotransfection of lenti- EGFP and lenti-Cre after 7 days, (Fig. 4B). EGFP efficiency treated with lenti-Cre was scored by FACS, (Fig. 4C). PCR of Genomic DNA demonstrated that EGFP was deleted by lenti-Cre, (Fig. 4D), and positive control was the result of RT-PCR of EGFP.
  • Fig. 5 lentiviral vector-mediated DOX-induced gene expression.
  • the EGFP was expressed in HAC based on PLVTHM, (Fig. 5A).
  • the HAC were cotransduced with LVtTR-KRAB and PLVTHM, (Fig. 5B), as well as LVtTR-KRAB and PLVTHM in the presence of DOX, (Fig. 5C). HAC were observed with a fluorescence microscope (x 10).
  • Fig. 6 the expression of viral protein GAG and EGFP in the HAC and HeIa cell cultured with HAC conditional medium.
  • RT-PCR of EGFP showed HAC transduced with lenti-EGFP sustain EGFP expression, but the HeIa cell cultured with HAC conditional medium has no EGFP expression, (Fig. 6A).
  • Structure gene GAG cannot be detected either in the lenti-EGFP transduced HAC or in the HeIa cell cultured with HAC conditional medium, (Fig. 6B).
  • Fig. 7 the average infarct volumes in the experimental groups. A reduction in the volume of ischemic damage was detected in the cell transplanted groups compared to the PBS group when measured at 16 days after ischemia.
  • FIG. 8 immunostaining of HAC-GDNF in vivo. 14 days after transplantation immunohistochemical staining of GDNF from a xenografted rat biain showed more GDNF positive HAC in the brain of the HAC-GDNF group, (Fig. 8A).
  • Fig.8B shows high-power photomicrographs depicting the boxed areas in Fig. 8A. DAB and hematoxylin counterstaining are shown in Fig. 8C and 8D. As shown, there was no nestin expression in the contralateral area.
  • Fig. 9A Fig. 9A
  • Fig. 9C DAB and hematoxylin counterstaining
  • Fig. 9B is the same site of contralateral tissue. DAB and hematoxylin counterstaining are shown in Fig. 9D.
  • Fig. 11 beam-walking test to detect coordination function.
  • the present invention provides a population of human amniotic cells comprising lentiviral vectors in which each lentiviral vector includes at least one exogenous gene element.
  • lentiviral vectors are agents that can transport a gene of interest into a cell without degradation in all cells.
  • Lentiviral vectors can also include a promoter yielding expression of a gene in cells such as, for example, amniotic cells.
  • Lentiviral vectors are based on the nucleic acid backbone of a virus from the lentiviral family of viruses.
  • a lentiviral vector can also comprise first, second and third generation lentiviruses.
  • lentiviral vectors can provide for gene transfer more efficiently than Ad and AAV. Moreover, lentiviral vectors can stably express a foreign transgene without detectable pathogenesis or irnmunogenicity from viral proteins. Human amniotic cells transduced by lentiviral vectors according to the invention can be particularly effective for gene therapy.
  • lentiviral vectors are third generation lentiviruses. For example, third generation lentiviruses have lentiviral packaging genes split into at least 3 independent plasmids.
  • lentiviral vectors such as third generation lentiviruses can lack the HW- I tat gene (a strong transcriptional activator of the HW-I LTR promoter essential for viral replication) and accessory genes (vpr, vpu, vi/and nef).
  • the EMV gene of HW-I can be replaced with the VSVG gene and the residual HIV genome may be divided into two expression constructs.
  • Exemplary lentiviruses can comprise a PWPT, PLVTHM or PLV vector.
  • HAC develop from early inner cell masses of blastula about 8 days after fertilization.
  • Human amniotic cells can be obtained from human amnion.
  • Exemplary HAC comprise amniotic epithelial and mesenchymal stem cells.
  • Human amniotic cells can express some markers normally present on stem cells such as, for example, GFAP, MAP2, nestin and AFP. Knezevic et al., Anat, 1996, 189: 1-7; Yuge et al, Transplantation, 2004, 77(9): 1452-4; Takashima et al., Cell Struct. Funct, 2004, 29(3): 73-84; Sakuragawa et al., Neurosci. Lett., 1996, 209(1): 9-12; Wei et al., Cell Transplant., 2003, 12(5): 545-52.
  • HAC HLA class Ib
  • Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources such as, for example, FKAi-inducible, Cre-loxP and doxycycline-inducible systems.
  • a vector for transduction of HAC can be designed to carry exogenous gene elements that may be expressed by the cells.
  • An exogenous gene element can, for example, comprise a marker gene.
  • Such a marker gene can produce a product and be used to determine whether a gene has been delivered to a cell and expressed thereby.
  • Exemplary marker genes can include the E. CoIi lacZ gene, which encodes P-galactosidase, green fluorescent protein (GFP) or the enhanced green fluorescent protein (EGFP).
  • exogenous gene elements can also include genes that are replacing or supplementing a native gene, which may be capable of treating a central nervous system disease.
  • the exogenous gene element can encode nerve growth factor, brain-derived neurotrophic factor, hypoxanthine guanine phosphoribosyltransferase, glia- derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, bcl-2, tetrahydrobiopterin synthase or combinations thereof.
  • Preferred exogenous gene elements can comprises glia-derived neurotrophic factor (BDNF) or brain-derived neurotrophic factor (GDNF) genes.
  • BDNF glia-derived neurotrophic factor
  • GDNF brain-derived neurotrophic factor
  • Neurotrophic factors are responsible for the growth and survival of nerve cells during development as well as the maintenance of adult nerve cells. For example, animal studies and in vitro models have shown that certain neurotrophic factors are capable of making damaged nerve cells regrow.
  • amniotic cells comprising lentiviral vectors in which the lentiviral vectors include at least one exogenous gene element that encodes for a neurotrophic factor.
  • Exemplary neurotrophic factors can be used to treat or reverse the effects of central nervous system diseases.
  • Brain-derived neurotrophic factor (BDNF) belongs to the neurotrophin family of trophic factors. BDNF is widely and abundantly expressed in the CNS and is available to some peripheral nervous system neurons that uptake the neurotrophin produced by peripheral tissues. BDNF promotes survival and differentiation of certain neuronal populations during development.
  • BDNF can modulate neuronal synaptic strength and has been implicated in hippocampal mechanisms of learning and memory as well as the spinal mechanisms for pain.
  • CNS disorders are also associated with a decrease in trophic support.
  • BDNF and its high affinity receptors are abundant throughout the whole CNS, BDNF can be a potent neuroprotective agent that is effective for the treatment of, without limitation, Parkinson's disease, Alzheimer's disease, depression, epilepsy and chronic pain.
  • Glial cell derived neurotrophic factor, GDNF also belongs to the family of neurotrophic factor proteins. GDNF enhances the survival and morphological differentiation of dopaminergic neurons and increases their uptake of dopamine.
  • GDNF can rescue motor neurons from programmed cell death and death caused by axotomy.
  • GDNF is a particularly potent factor for survival and axonal growth of mesencephalic dopaminergic neuron and has been shown to ameliorate motor deficits and reduce brain damage in several animal models.
  • exogenous gene element comprises the GDNF gene.
  • Figure 1 shows lentiviral constructs that comprise a GDNF exogenous gene element.
  • Exemplary exogenous gene elements such as those described herein can comprise sequences obtained from, without limitation, the references provided herein and GenBank.
  • the exogenous gene elements can be constructed or modified through conventional recombinant techniques.
  • HAC comprising lentiviral vectors with exogenous gene elements and compositions thereof can be transplanted into the body of a subject for gene therapy.
  • the HAC comprising lentiviral vectors with exogenous gene elements can be used in the therapy of cerebral nervous system diseases.
  • Diseases of the central nervous system include disorders of the brain, spinal cord, cranial nerves, nerve roots and autonomic nervous system.
  • Central nervous system (CNS) diseases can also comprise, for example, cerebral ischemia, cerebral hemorrhage, central nervous system trauma, hereditary diseases of the nervous system, neurodegenerative diseases, spinal cord trauma, Parkinson's disease and neoplasms of the central nervous system.
  • Cerebral ischemia is an ischemic condition in which the brain or parts thereof do not receive enough blood flow to maintain normal neurological function.
  • the condition can be the result of various diseases or arterial obstructions such as strangulation. Cerebral ischemia can bring about neuron death and induce brain disorder.
  • cerebral hemorrhage is a sudden loss of consciousness resulting from the rupture or occlusion of a blood vessel, leading to oxygen lack in the brain.
  • CNS trauma is damage to the brain and spinal cord that results from direct injury to them or from indirect injury due to damage of the bones, soft tissues or blood vessels surrounding the brain and spinal cord.
  • Hereditary diseases of the nervous system are a group of inherited, slowly progressive disorders that can result from progressive damage to nerves such as, for example, Huntington's chorea (HD), amaurotic family idiocy (Tay-Sachs), dentatorubropallidoluysian atrophy (DRPLA) and Machado-Joseph disease (MJD).
  • a neurodegenerative disease is a disorder caused by the deterioration of certain nerve cells (neurons). Changes in these cells can cause them to function abnormally and eventually bring about their death.
  • Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis (ALS) as well as multiple sclerosis are due to neuronal degeneration in the central nervous system.
  • Neoplasms of the central nervous system include, without Imitation, the cerebral hemispheres, basal ganglia, hypothalamus, thalamus, brain stem and cerebellum.
  • Brain neoplasms can be subdivided into primary (originating from brain tissues) and secondary (metastatic) forms.
  • Primary neoplasms can also be subdivided into benign and malignant forms.
  • brain tumors may be classified by age of onset, histologic type or presenting locations.
  • Spinal cord trauma is damage to the spinal cord that results from direct injury to the cord itself or from indirect injury due to damage to the bones, soft tissues or blood vessels surrounding the spinal cord.
  • Parkinson's disease is a chronic progressive nervous disease that is linked to decreased dopamine production in the substantia nigra and can be marked by tremors and weakness in resting muscles as well as by a shuffling gait.
  • the symptoms of Parkinson's disease are caused by a loss of nerve cells in a part of the brain called the substantia nigra, resulting in a decrease in dopamine (a neurochemical) throughout the brain. This destruction occurs due to genetic or environmental factors as well as combinations thereof.
  • intracerebral grafting of GDNF-transduced HAC into ischemic rats prepared by middle cerebral artery occlusion (MCAo) can significantly ameliorate behavioral dysfunctions and reduce infarct volumes.
  • MCAo middle cerebral artery occlusion
  • neuronal markers and markers of neuronal stem cells can be detected in these transplanted HAC.
  • a number of these transplanted HAC survive and migrate to the infarct area.
  • the BDNF gene which is an important member of the growth factors for the nervous system, plays a physiological role in the development of the central nervous system and regulation of adult neurons.
  • the BDNF gene also provides for potent neuroprotective effects on a variety of damaged neurons (irrespective of the reasons for damage).
  • the BDNF protein of AD or PD subjects is less than that of those that are normal.
  • Intracerebral grafting of BDNF-transduced HAC (for example, epithelial) into a PD subject (rat) can significantly ameliorate behavioral dysfunction and increase the BDNF protein.
  • grafting of BDNF-transduced HAC into a Rhesus monkey with incomplete dorsal spinal cord injury can also significantly ameliorate behavioral dysfunction.
  • pharmaceutically acceptable carriers may include sterile aqueous solutions, suspensions, and emulsions.
  • Aqueous solution carriers can also include, without limitation, PBS or Hankas' solutions.
  • Exemplary emulsions or suspensions can include collagen, hydroxyproxyl cellulose, niicrocrystalline cellulose, amylum, PVP, agar, pectin, magnesium aluminate silicate or magnesium aluminate.
  • the number of cells to be transplanted can be appropriately determined depending on the conditions of the patient and on the ability to produce the desired gene product of the cells according to the invention. In one embodiment, the number of cells transplanted can be about 10 5 to 10 10 .
  • Therapy methods can also refer to a method of transplantation of cells and compositions thereof. Moreover, therapy methods may also include sterile methods such as, for example, direct injection or encephalic transplantation.
  • sterile methods such as, for example, direct injection or encephalic transplantation.
  • EXAMPLE I Culturing of HAC An amnion membrane was mechanically peeled from the chorion of a placenta and was extensively scraped out to remove the underlying tissues (the spongy and fibroblast layers) to obtain a pure epithelial layer with basement membrane. The membrane was cut with a razor to yield a segment. Enough enzyme solution to obtain a signal cell was added. HAC were then cultured in a cell culture medium.
  • lentiviral vectors were produced by transient transfection of 293T cells. 20 ⁇ g of lentiviral vectors (PWPT, LVTHM or PWPT- GDNF, Fig. 1), 10/ig of pMDlg/pRRE (or pCMVdR 8.2), 5 ⁇ g of pMD2 G and 5 ⁇ g of pRSV- REV were mixed and adjusted to 250 ⁇ l with water.
  • the dishes were placed in a 37°C humidified incubator with a 5 % CO 2 atmosphere. The medium was aspirated. 14 hours later, 10 ml of fresh DMEM-10 % FBS (PAA Austria) prewarmed to 37°C was gently added, followed by incubation for 28 hours. The virus was collected and cleared via centrifugation at 1500 rpm for 15 minutes and filtered through a 0.45 ⁇ m filter. Ultracentrifugation occurred for 90 minutes at 80,000 g and 4 0 C. An aliquot of the supernatant was resuspended as pellets with 1 ml PBS and stored at -8O 0 C. Titration of the concentrated supernatants was performed by serial dilutions of vector stocks on 1 ⁇ 10 HeIa cells, followed by fluorescence-activated cytometric, Beckton Dickinson
  • the viral vector was added to cultured amniotic cells on the following day for 2 days in the presence of 1-10 ⁇ g/ml polybrene (Sigma-Aldrich). After washing with PBS, HAC continued to be cultured in a cell culture medium such that exogenous gene elements could be introduced into HAC.
  • polybrene Sigma-Aldrich
  • amnion membrane was mechanically peeled from the chorion of a placenta obtained from a woman with an uncomplicated cesarean section and was extensively scraped out to remove the underlying tissues (the spongy and fibroblast layers) to obtain a pure epithelial layer with basement membrane.
  • the membrane was placed in a 250 ml wide-mouthed flask containing a RPMI 1640 medium and cut with a razor to yield a 0.5-1.0 segment. Enough 37°C trypsin/EDTA solution was added to the culture to cover the membrane twice, the first and second times for about 30 and 15 minutes, respectively.
  • the obtained cells were seeded within six well plates in a RPMI-1640 medium supplemented with 10 % fetal calf serum (PAA Austria), streptomycin 100 ⁇ g/ml, penicillin 100 U/ml and glutamine 0.3 mg/ml, followed by incubation under a humidified atmosphere of 5 % CO 2 in air at 37°C.
  • HAC were plated on a 6-well plate (2 x 10 s cells/well, Costar) and polybrene (Sigma-Aldrich) was added to the wells for a final concentration of 8 ⁇ g/ml.
  • DOTAP Boehringer Mannheim
  • Transduced HAC was cultured for 1 week.
  • EGFP expression was visualized by fluorescent microphotograph and analyzed by fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • Genomic DNA was isolated as described by, for example, Promega and total RNA isolation and reverse transcription were performed (Promega).
  • FACS fluorescence-activated cell sorting
  • Primer sequences were EGFP (upstream 5'-cgagctggacggcgacgtaaac-3' and downstream 5'-gcgcttctcgttggggtcttg-3') and ⁇ -actin (upstream 5'-aacgagcggttccgatgccctgag-3' and downstream 5'-tgtcgccttcaccgttccagtt-3').
  • Amplification conditions were 94 0 C for 5 minutes, 28 cycles of 94°C for 1 minute, 58 0 C for 1 minute, 72 0 C for 1 minute and 72°C for 10 minutes.
  • the expected length of the EGFP RT-PCR products was 597 bp.
  • the expression difference was normalized by the respective ⁇ - actins (590 bp).
  • Ten microliters from each RT-PCR product were loaded on a 1.5 % agarose gel containing 0.5 ⁇ g/ml of ethidium bromide and separated by electrophoresis.
  • EGFP can be observed with fluorescence microscopy in the 4th day after transduction.
  • HAC was transduced with DOTAP and these cells were scored by fluorescence- activated cell sorting (FACS) in the 7th day.
  • FACS fluorescence- activated cell sorting
  • Transduction of lentiviral vectors was detected to be more efficient than DOTAP with about 90 % of the cells successfully transduced with lentiviral vectors, (Fig. 2A), and less than 5 % with DOTAP (not shown).
  • EGFP expression was measured for 5 weeks in succession and EGFP-positive HAC were maintained during the culture period, (Fig. 2B).
  • lentiviral vector transduced HAC of the invention can provide for steady gene transmission, which may be useful in the prophylaxis or treatment of CNS diseases.
  • HAC human immunodeficiency virus
  • pLVTHM and pLVTHMsiGFP lentiviral vectors
  • MOI 100
  • FACS fluorescence-activated cell sorting
  • Primer sequences were EGFP (upstream 5'-cgagctggacggcgacgtaaac-3' and downstream 5'- gcgcttctcgttggggtcttg-3') and ⁇ -actin (upstream 5'-aacgagcggttccgatgccctgag-3' and downstream 5'-tgtcgccttcaccgttccagtt-3').
  • Amplification conditions were 94°C for 5 minutes, 28 cycles of 94°C for 1 minute, 58°C for 1 minute, 72°C for 1 minute and 72°C for 10 minutes.
  • the expected length of the EGFP PCR products was 597 bp.
  • the expression difference was normalized by the respective ⁇ -actins (590 bp). Ten microliters from each PCR product were loaded on a 1.5 % agarose gel containing 0.5 ⁇ g/ml of ethidium bromide and separated by electrophoresis.
  • RNA interference was the best gene-silencing pathway of RNA mediated gene regulation in post-transcription.
  • Expression of siRNAs delivered by Jentiviral vectors can be used to functionally silence EGFP expression in HAC.
  • a sequence of siGFP in the lentiviral vector (PLVTHMsiGFP) was transcripted into dsRNA, which mediates sequence specific cleavage with the formation of a ribonucleoprotein complex, hi the first experiment, the ability of lentiviral vector PLVTHM to express EGFP in HAC, (Fig. 3A), was probed.
  • MOI 100 ensured good rates of transduction.
  • the HAC-EGFP cells transduced with PLVTHM had strong EGFP expression irrespective of culture conditions.
  • lentiviral vectors PWPT and PWPT-Cre
  • MOI 100
  • Transduced HAC were cultured for 1 week.
  • EGFP expression was visualized by fluorescent microphotograph and analyzed by fluorescence-activated cell sorting (FACS). Genomic DNA was isolated as described by, for example, Promega.
  • FACS fluorescence-activated cell sorting
  • Primer sequences were EGFP (upstream 5'-cgagctggacggcgacgtaaao-3' and downstream 5'-gcgcttctcgttggggtcttg-3') and ⁇ -actin (upstream 5'-aacgagcggttccgatgccctgag-3' and downstream 5'-tgtcgccttcaccgttccagtt-3').
  • Amplification conditions were 94°C for 5 minutes, 28 cycles of 94°C for 1 minute, 58°C for 1 minute, 72 0 C for 1 minute and 72°C for 10 minutes.
  • the expected length of the EGFP PCR products was 597 bp.
  • the expression difference was normalized by the respective ⁇ -actins (590 bp). Ten microliters from each PCR product were loaded on a 1.5 % agarose gel containing 0.5 ⁇ g/ml of ethidium bromide and separated by electrophoresis.
  • Cre is a member of the integrase family of site-specific recombinases that catalyzes recombinationbetween loxP DNA elements. Sternberg et al., J. MoI. Biol., 1981, 150: 467-86. An attempt was made to create a line of HAC in which Cre catalyzed loxP-flanked specific gene deletions based on lentiviral vectors. For example, there is a loxP in the 3'LTR of lentiviral vectors and, after reverse transcription, loxP integrated into both ends of LTR. Cre catalyzed the deletion of loxP-flanked lentiviral fragments in the genome of HAC.
  • tTR-KRAB binds specifically to tetO, providing a means for suppression of the activity of the nearby promoter (up to 3 kb from its DNA-binding site).
  • tTR-KRAB was sequestered away from tetO, thereby permitting gene expression. Lentiviral vector mediated doxycycline-induced system was tested for the doxycycline induced regulation of EGFP in HAC, after EGFP was observed in PLVTHM transduced HAC, (Fig. 5A).
  • tTRKRAB based on lentiviral vectors suppressed the expression of EGFP in the absence of doxycycline, (Fig. 5B).
  • addition of doxycycline to the dually transduced cells resulted in EGFP (re)expression, (Fig. 5C).
  • the cells were transduced with PWPT for 3 days and washed intensively. Thereafter, the medium was collected and filtered through a 0.45 ⁇ m filter. HeIa cells with the medium were cultured for 3 days and genomic DNA of the HeIa cells and HAC were isolated as described by, for example, Promega.
  • Genomic DNA (100 ng) was then subjected to PCR using primers EGFP (upstream 5 '— cgagctggacggcgacgtaaac-3' and downstream 5'-gcgcttctcgttggggtcttg-3') and ⁇ -actin (upstream 5'-aacgagcggttccgatgccctgag-3' and downstream 5'-tgtcgccttcaccgttccagtt-3') as well as primers homologous to the HIV-I gag gene (upstream 5'-gagtatctgatcatactgtcctac-3' and downstream 5'-ggaactactagtacccttcaggaa-3').
  • primers EGFP upstream 5 '— cgagctggacggcgacgtaaac-3' and downstream 5'-gcgcttctcg
  • Amplification conditions were 94°C for 5 minutes, 28 cycles of 94°C for 1 minute, 58 0 C for 1 minute, 72°C for 1 minute and 72°C for 10 minutes.
  • the expected length of EGFP and gag products was 597 bp and 912 bp, respectively.
  • the expression difference was normalized by the respective ⁇ -actins (590 bp).
  • Ten microliters from each RT-PCR product were loaded on a 1.5 % agarose gel containing 0.5 ⁇ g/ml of ethidium bromide and separated by electrophoresis.
  • Lenti-GDNF 50 was added to DMEM-F 12 cultured HAC on the following day for 2 days in the presence of 8 ⁇ g/ml polybrene (Sigma-Aldrich). After growing for 5 days in a DMEM-F 12 medium without fetal calf serum, HAC were washed with PBS and then incubated in 0.25 % trypsin (Sigma)/0.05 % DNase I (Sigma)/PBS at 37°C for 20 minutes. The cells were rinsed 2-3 times with 0.05 % DNase I/PBS and mechanically dissociated into a single cell suspension. An aliquot of the cell suspension was assessed with regard to cell viability (trypan blue) and concentration.
  • the viability of the cell suspension prior to grafting was more than 95 %.
  • 8 x 10 5 GDNF modified HAC (HAC-GDNF) in 5 ⁇ l of PBS were injected into the right dorsolateral striatum of MCAo rats.
  • HAC-GDNF GDNF modified HAC
  • Three rats of each group were anesthetized and sacrificed with excess phenobarbital 16 days after the surgery. Their brains were carefully removed and sliced to 2 mm slices using a mold. The slices were stained with 2,3,5-triphenyltetrazolium chloride (TTC, 2 % solution in PBS) for 30 minutes. The slices were also photographed with, an image acquirement (Leica) system. Image analysis software AutoCAD (AutoDesk) was used for an estimation of the infracted volume. The results were expressed as a percentage of the hemisphere.
  • the slides were incubated for 48 hours at 4°C with a first antibody specific against MAP2 (1 :200, Sigma M4403) and GDNF (1 : 100, Santa Cruz SC- 9010).
  • the sections were then rinsed 3 times in PBS (pH 7.4), followed by biotin conjugated antimouse or antirabbit IgG (Vector Laboratories). Thereafter, sections were washed in PBS and incubated with an avidin-biotin-horseradish peroxidase complex.
  • the preparations were also stained using a Vectastain ABC kit (Vector Laboratories).
  • the slide was colorized with diaminobenzidine (DAB). The immunohistochemical studies were repeated at least three times.
  • HAC-GDNF GDNF engineered HAC
  • the neurological findings were scored on a modified scoring system that was developed by Longa et al. For example, a score of O indicates no neurological deficits, 1 indicates that the rat had difficulty in fully extending the contralateral forelimb, 2 indicates that the rat could not extend the contralateral forelimb, 3 indicates a mild circling to the contralateral side, 4 indicates a severe circling to the contralateral side and 5 indicates falling to the contralateral side.
  • the severity of neurological deficits was observed in the three stroke groups. Treatment was examined and analyzed statistically by mean ⁇ SE with a significance level ofP ⁇ 0.05.
  • Rats in the stroke groups were examined at 4 time points after cell transplantation, up to 16 days.
  • the beam-walking test was described by Ohlson et al.
  • the beam was 1750 mm long and 19 mm wide.
  • the beam was placed 700 mm above the floor.
  • a wall was alternately placed 2 cm near the beam (rats are more willing to walk when a wall is placed next to the beam). Scoring was from 0 to 6.
  • the rat falls down, for 1, the rat is unable to traverse the beam, but remains sitting across the beam, for 2, the rat falls down while walking, for 3, the rat can traverse the beam, but the affected hindliirib does not aid in forward locomotion, for 4, the rat traverses the beam with more than 50 % footslips, for 5, the rat crosses the beam with a few footslips and for 6, the rat crosses the beam with no footslips. When the rat walked on the beam, such scoring was conducted. Treatment was examined and analyzed statistically by mean ⁇ SE with a significance level of P ⁇ 0.05.
  • HAC produced GDNF can rapidly rescue the deficits of a subject after MCAo and HAC also have a significant role in the following recovery period.
  • neurotrophic factors or anti-inflammation factors secreted by HAC may be caused by the neurotrophic factors or anti-inflammation factors secreted by HAC as well as differentiation to neuronal cells.
  • HAC-GDNF were prepared and suspended in PBS.
  • Experimental PD was produced in adult rats with the intracerebral injection of a neurotoxin, 6-hydroxydopamine.
  • the toxin was injected in the medial forebrain bundle of one side of a rat brain under stereotaxic guidance.
  • the subjects were tested with apomorphine, which causes aberrant rotation behavior in recipients with a successful biochemical lesion of the nigral-striatal tract.
  • PLVTHM-BDNF 80 was added to DMEM-F12 cultured cells on the following day for 2 days in the presence of 8 ⁇ g/ml polybrene (Sigma-Aldrich). After growing for 5 days in a DMEM-F12 medium without fetal calf serum, HAC were washed with PBS and then incubated in 0.25 % trypsin (Sigma)/0.05 % DNase I (Sigma)/PBS at 37°C for 20 minutes. The cells were rinsed 2-3 times with 0.05 % DNase I/PBS and mechanically dissociated into a single cell suspension. The cell concentration was adjusted to 1-2 x 10 8 .
  • a rhesus monkey with incomplete dorsal spinal cord injury was studied according to the Tator method. Basso et al., J. Neurotrauma, 1995,12(1): 1-21.
  • 1 x lO 7 BDNF modified HAC were injected into the injured position of 2 monkeys.
  • PBS was injected into the injured position of one monkey. Two months later, a BBB locomotor rating scale was evaluated. Basso et al., J. Neurotrauma, 1995,12(1): 1-21.
  • the BBB score of the cell transplantation group was 9.7.
  • the BBB score of the control group was 5.2.
  • the HAC infected with PLVTHM-BDNF were shown to improve hindlimb motor function of the subject.

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  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Reproductive Health (AREA)
  • Epidemiology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Psychology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Gynecology & Obstetrics (AREA)
  • Plant Pathology (AREA)
  • Psychiatry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)

Abstract

L'invention concerne une population de cellules amniotiques humaines comprenant des vecteurs lentiviraux qui contiennent au moins un élément génétique exogène et des méthodes permettant de fabriquer ces cellules. Les cellules selon l'invention peuvent être utilisées pour traiter des maladies telles que des maladies liées aux troubles neurologiques, par exemple, l'ischémie cérébrale, la maladie vasculaire cérébrale, les lésions du système nerveux central, les maladies génétiques du système nerveux, les maladies dégénératives du système nerveux cérébral, les tumeurs du système nerveux cérébral et des combinaisons de ces dernières.
EP06753014A 2005-06-24 2006-06-23 Cellules amniotiques et leurs méthodes d'utilisation Withdrawn EP1893750A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNA2005100271288A CN1884495A (zh) 2005-06-24 2005-06-24 可表达外源性基因的人羊膜细胞及其制备方法和应用
PCT/CN2006/001437 WO2006136114A1 (fr) 2005-06-24 2006-06-23 Cellules amniotiques et leurs methodes d'utilisation

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EP1893750A1 true EP1893750A1 (fr) 2008-03-05
EP1893750A4 EP1893750A4 (fr) 2009-05-13

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EP (1) EP1893750A4 (fr)
JP (1) JP2008543319A (fr)
KR (1) KR20080025173A (fr)
CN (1) CN1884495A (fr)
AU (1) AU2006261485A1 (fr)
CA (1) CA2620946A1 (fr)
WO (1) WO2006136114A1 (fr)

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Publication number Priority date Publication date Assignee Title
GB0514567D0 (en) * 2005-07-15 2005-08-24 Univ Nottingham Surgical membrane
EP2067858A1 (fr) * 2007-12-07 2009-06-10 Universidad de Sevilla Modèles d'animaux pour maladies neurodégénératives
CN101721430B (zh) * 2008-10-22 2013-01-23 中美赛傲(上海)生物技术有限公司 羊膜上皮细胞在制药中的用途
CN103705979B (zh) * 2013-09-18 2016-10-05 中国人民解放军海军总医院 用于神经修复的功能组织工程材料及其制备与用途

Citations (2)

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WO2003056022A2 (fr) * 2001-12-21 2003-07-10 Oxford Biomedica (Uk) Limited Organisme transgenique
WO2004000164A2 (fr) * 2002-06-24 2003-12-31 Amniotech, Inc. Liberation de molecules bioactives mediee par la membrane amniotique

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JP3806189B2 (ja) * 1995-07-27 2006-08-09 株式会社エスアールエル ヒト羊膜細胞への遺伝子導入方法及び遺伝子治療用細胞の調製方法
JP4217037B2 (ja) * 2001-08-10 2009-01-28 宣男 櫻川 ヒト羊膜間葉細胞由来ヒト神経幹細胞

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003056022A2 (fr) * 2001-12-21 2003-07-10 Oxford Biomedica (Uk) Limited Organisme transgenique
WO2004000164A2 (fr) * 2002-06-24 2003-12-31 Amniotech, Inc. Liberation de molecules bioactives mediee par la membrane amniotique

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Title
BUCHSCHACHER G L ET AL: "DEVELOPMENT OF LENTIVIRAL VECTORS FOR GENE THERAPY FOR HUMAN DISEASES" BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 95, no. 8, 1 January 2000 (2000-01-01), pages 2499-2504, XP000916373 ISSN: 0006-4971 *
KOSUGA MOTOMICHI ET AL: "Phenotype correction in murine mucopolysaccharidosis type VII by transplantation of human amniotic epithelial cells after adenovirus-mediated gene transfer" CELL TRANSPLANTATION, vol. 9, no. 5, September 2000 (2000-09), pages 687-692, XP009114244 ISSN: 0963-6897 *
LIU TIANJIN ET AL: "Human amniotic epithelial cells ameliorate behavioral dysfunction and reduce infarct size in the rat middle cerebral artery occlusion model." SHOCK (AUGUSTA, GA.) MAY 2008, vol. 29, no. 5, May 2008 (2008-05), pages 603-611, XP009114219 ISSN: 1073-2322 *
See also references of WO2006136114A1 *
TAKAHASHI SATORU ET AL: "A novel approach to ex vivo gene therapy for familial hypercholesterolemia using human amniotic epithelial cells as a transgene carrier" TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, vol. 193, no. 4, April 2001 (2001-04), pages 279-292, XP002520718 ISSN: 0040-8727 *

Also Published As

Publication number Publication date
KR20080025173A (ko) 2008-03-19
CA2620946A1 (fr) 2006-12-28
AU2006261485A1 (en) 2006-12-28
EP1893750A4 (fr) 2009-05-13
JP2008543319A (ja) 2008-12-04
CN1884495A (zh) 2006-12-27
WO2006136114A1 (fr) 2006-12-28

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