CA2620946A1 - Amniotic cells and methods for use thereof - Google Patents

Amniotic cells and methods for use thereof Download PDF

Info

Publication number
CA2620946A1
CA2620946A1 CA002620946A CA2620946A CA2620946A1 CA 2620946 A1 CA2620946 A1 CA 2620946A1 CA 002620946 A CA002620946 A CA 002620946A CA 2620946 A CA2620946 A CA 2620946A CA 2620946 A1 CA2620946 A1 CA 2620946A1
Authority
CA
Canada
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.)
Abandoned
Application number
CA002620946A
Other languages
French (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
Original Assignee
CELL STAR BIO-TECHNOLOGIES Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by CELL STAR BIO-TECHNOLOGIES Co Ltd filed Critical CELL STAR BIO-TECHNOLOGIES Co Ltd
Publication of CA2620946A1 publication Critical patent/CA2620946A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48Reproductive organs
    • A61K35/50Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10Cells modified by introduction of foreign genetic material
    • C12N5/12Fused cells, e.g. hybridomas
    • C12N5/16Animal cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18Growth factors; Growth regulators
    • A61K38/185Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00Drugs for dermatological disorders
    • A61P17/02Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/14Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16Anti-Parkinson drugs
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00Drugs for disorders of the nervous system
    • A61P25/28Drugs 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • A61P9/10Drugs 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66General 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • C12N5/0603Embryonic cells ; Embryoid bodies
    • C12N5/0605Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00Genetically modified cells
    • C12N2510/02Cells for production
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00Uses of viruses
    • C12N2799/02Uses of viruses as vector
    • C12N2799/021Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/001Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/005Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB
    • C12N2830/006Vector systems having a special element relevant for transcription controllable enhancer/promoter combination repressible enhancer/promoter combination, e.g. KRAB tet repressible

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • 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

The present invention provides a population of human amniotic cells comprising lentiviral vectors with at least one exogenous gene element and methods for making such cells. The cells of the invention can be used for treating diseases such as those related to neurologic disorders including, for example, cerebral ischemia, cerebral vascular disease, central nervous system injuries, genetic diseases of the nervous system, degenerative diseases of the cerebral nervous system, tumors of the cerebral nervous system and combinations thereof.

Description

AMNTOTIC CELLS AND METHODS FOR USE THEREOF

FIELD OF THE INVENTION

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 malcing such cells and methods for treating diseases.

RACKGROUND OF THE INVENTION

Humans can suffer from such 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, Parlc.inson's disease, Huntington's disease and Alzheimer's disease) and tumors of the cerebral nervous system. To date, there are few effective therapeutic treatinents or drugs for central nervous system diseases. Typically, the death and cripple rates for central nervous system diseases are very high. For example, every year 16,400,000 people suffer from brain strokes, brain injuries and spinal cord injuries throughout the world. Moreover, 4,100,000 of these people die each year due to such maladies.
Recently, gene therapy has developed as a potentially potent method for treating many neurological diseases previously considered refractory to conventional approaches. In general, 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. For example, mutation recessive genes of central nervous system diseases express proteins and gene therapy can be used to down-regulate expression of theses proteiuls via antisense or RNAi to the mutated mRNA. Another strategy is to compensate for loss-funetion enzymes or proteins of the brain by transferring genes. Lastly, neurons can be protected by delivery of proteins such as growth factors, antioxidants, HSP or anti-apoptotic molecules. Natsume et al., Exp. Neurol., 2001, 169: 231-33; Berry et al., Curr. Opin. Mol.
Ther., 2001, 3: 338-49.

Five types of virus vectors 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. At present, there are many issues relating to efficacy, stability, regulatability and safety of directly using genetically-engineered virus vectors for in vivo gene therapy. These issues include, for example, the stability and specificity of virus vectors in vivo. Alternatively, using embryonic or somatic stem cells for ex vivo gene tlierapy poses concerns relating to safety, inunune rejection, cell sources and ethical implications. Georgievska et al., Eur. J. Neurosci., 2004, 20(11):
3121-30; Englund et al., Exp. Neurol., 2002, 173(1): 1-2; Kahn et al., Blood, 2004, 103(8):
2942-9; De Pa1ma et al., Nat. Med., 2003, 9(6): 789-95; Imren et al., J. Clin. Invest., 2004:
953-62.

SUMMARY OF THE INVENTION

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.
Preferably, the exogenous gene elements of the vector are capable of being expressed by the cells.
Another aspect 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. For example, 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 amn.iotic cells, which can be useful for gene therapy. Preferably, the exogenous gene element can be highly expressed in the humasi 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 systenl 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. In one embodiment, human amniotic cells according to the invention can minimize or eliminate rejection reactions or topical 'uiflammations. Prior to transduction, the human amniotic cells can be derived from, without limitation, a donor, tissue-cultures or subject (for example, patient).
In one embodiment, 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.
Generally, "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.
For example, 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 may be useful in the treatment or prophylaxis of, for example, CNS diseases. In one embodiment, a method is provided 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.
Preferably, lentiviral vector transduced HAC of the invention can be adininistered to a patient in an amount or dosage suitable for treating a CNS disease. Generally, 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 lentiviral vector transduced HAC of the invention to a subject may be local or systemic and accomplished intravenously, intraarterially, intrathecally (via the spinal fluid) or the like.
Administration may also be intradennal or intracavitary, depending upon the body site wider therapy.
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 lentiviral 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. As described, administration of lentiviral 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 lentiviral vector transduced HAC to a subject can be by a general or local administration route. For example, the lentiviral vector transduced HAC or compositions thereof may be administered to the patient such that it is delivered throughout the body. Alternatively, the lentiviral 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 lentiviral 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.
Remington's Pharmaceutical Sciences, 15th Edition, Mack Publishing Co., 1975: 1405-1487;
The National Formulary XIV., 14th Edition, American Pharmaceutical Association, 1975.
Exemplary pharmaceutically acceptable carriers for lentiviral 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. In one embodiment, lentiviral 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 .4 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.
In one embodiment, 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. For example, 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 ainount" 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.
Another exatn.ple of an effective anzount includes amounts or dosages that are capable of minimizing or preventing neuronal degeneration. When desired, 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).
A person of ordinary skill in the art can readily deterlnine an effective amount of lentiviral vector transduced HAC or compositions thereof by simply administering the cells or composition to a subject in increasing amounts over a period of time until effects of a disease are lessened. The determination of an effective amount for a particular subject is well known to those of ordinary skill in the art. The invention also contemplates that lentiviral vector transduced HAC can be used for ex vivo or in vivo gene therapy.
Preferably, for in vivo gene therapy of a CNS disease, retrovirus, AAV, lentiviral, pseudotyped lentiviral or Ad vectors for the transduction of HAC can be administered to a subject in an effective amount. These vectors can be administered in a composition S ~

comprising a pharmaceutically acceptable carrier. Moreover, 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. As described, administration can also be local or systemic and accomplished intravenously, intraarterially, intrathecally (via the spinal fluid) or via a graft.
In addition to using promoters to drive expression in HAC, an enhancer sequence may be used to increase the level of expression. Armelor et al, Proc. Natl. Acad.
Sci., 1973, 70:
2702. 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).
The invention also contemplates analogs, homologs, derivatives and variants of nerve growth factor, brain-d.erived neurotrophic factor, hypoxanthine guanine phosplloribosyltransferase, glia-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L-amino acid decarboxylase, bcl-2 or tetrahydrobiopterin synthase. Similarly, the invention contemplates analogs, hoinologs, 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.
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.
Additionally, the invention contemplates transduction of HAC via, without limitation, one or more retrovirus, AAV, lentiviral, pseudotyped lentiviral, Ad vectors and combinations thereof. For example, these vectors can be used either sequentially or in combination to transduce HAC. A vector can also comprise one or more exogenous elements.
In one embodiment, a population of human aniniotic cells comprising one or more lentiviral vectors is provided. For example, 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. Preferably, 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.
Moreover, a method of transducing a population of human amniotic cells with one or more lentiviral vectors is provided. Preferably, the method comprises culturing the human amniotic cells. For example, 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. In one embodiment, 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.
In one embodiment, a method for treating central nervous systein diseases in a subject is provided. Preferably, the method involves the use of a population of hunian amniotic cells comprising a lentiviral vector with an exogenous gene element. Exemplary 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. Different types of 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. Moreover, other murine retroviral vectors that can be used include those based on murine embryonic stenz cell virus (MESV) and murine steni 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.
Alternatively, 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.
Examples of promoters useful in the invention include constitutive and inducible promoters. Without limitation, such promoters can comprise CMV, SV40, retroviral LTR, EF, tetracycline inducible, inflammation induced, TNF-a and IL-1 promoters. In addition to HAC, the invention may comprise various other components and means to facilitate the delivery of cells to a subject. For example, 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. In one embodiment, the invention contemplates buffers such as, without limitation, HEPES, PBS or citrate-based buffers.
Furthermore, 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.
In one embodiment, exogenous elements can include, without limitation, growth factors, anti-microbial proteins, anti in.flamniatory 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. Moreover, examples of anti-microbial proteins can include bactericidallpermeability-increasing proteins, defensin, collectin, granulysin, protegrin-1, SMAP-29, lactoferrin and calgranulin C. Preferably, examples of anti-inflammatory proteins can include interleukin-1 receptor antagonists, interleukin-10, soluble TNF receptors and soluble CTLA4.
Protease inhibitors can also include, for example, TIlVIP-1, -2, -3, -4, PAI-1, PAI-2 and ecotin.
The invention also contemplates that 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. In one embodiment, 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, esopliagus, 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 marlcer sucll as, for example, a green fluorescent protein (GFP).
In one embodiment, 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. For example, a graft composition can also comprise a biocompatible matrix. The HAC of a graft composition can be talcen 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.
Preferably, a graft composition can be administered, without limitation, via the gastrointestinal tract (orally or as a suppository), parentexally (intramuscular, intravenous or subcutaneous) or topically.
The invention also contemplates composition comprising vector transduced HAC
and additional components. For examples, such components can include cells, genetically inodified cells, proteins, peptides, non-protein bioorganic substances, therapeutic agents (antiinflammatories, antibiotics, antivirals, antineoplastics or antimycotics) and combinations thereof. In one embodiment, 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 103 cells to about 1010 cells per day. The specific dosage used, however, 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 slcill in the art.

DESCRIPTION OF THE DRAWINGS
Other features and advantages of the invention may also be apparent from the following detailed description thereof, taken in conjunction with the accompanying drawings.
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. All vectors contain promoters:
elongation factor 1-alfa promoter (EF1-a) or human cytomegalovirus (CMV), which are robust transcriptional elements in most cell types.
Fig. 2, efficient transfer, integration and sustained long-term expression of EGFP in HAC. EGFP expression in HAC at 7 days after transduction with lentiviral vectors or DOTAP, (Fig. 2A). EGFP was evaluated on photographs that were talcen with a fluorescence microscope (x 10) and transduction efficiency was measuxed 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 sanze 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 ICRAB 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 Hela cell cultured with HAC conditional medium. RT-PCR of EGFP showed HAC transduced with lenti-EGFP sustain EGFP expression, but the Hela 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 Hela cell cultured with HAC
conditional medium, (Fig. 6B).
Fig. 7, the average iuifarct 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 ischeniia.
Fig. 8, immunostaining of HAC-GDNF in vivo. 14 days after transplantation immunohistochemical staining of GDNF from a xenografted rat brain showed more GDNF
positive HAC in the brain of the HAC-GDNF group, (Fig. 8A). Fig.BB 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. 9, 3 weeks after transplantation, strong MAP2 expression was detected in the injection tract with immunohistochemistry, (Fig. 9A), and DAB and hematoxylin counterstaining, (Fig. 9C). Fig. 9B is the same site of contralateral tissue.
DAB and hematoxylin counterstaining are shown in Fig. 9D.
Fig. 10, neurological test.
Fig. 11, beam walking test to detect coordination function.
DETAI_LED DESCRIPTION OF THE IWENTION
The present invention may be understood more readily by reference to this detailed description as well as the drawings and examples herein. The invention provides a population of human amniotic cells comprising lentiviral vectors in which each lentiviral vector includes at least one exogenous gene element. As used herein, 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 fainily of viruses. A lentiviral vector can also comprise first, second and third generation lentiviruses. In one embodiment, 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 irnrnunogenicity from viral proteins. Human amniotic cells transduced by lentiviral vectors according to the invention can be particularly effective for gene therapy.
In one embodiment, lentiviral vectors are third generation lentiviruses. For example, third generation lentiviruses have lentiviral packaging genes split into at least 3 independent plasnmids. Moreover, lentiviral vectors such as third generation lentiviruses can lack the HN-1 tat gene (a strong transcriptional activator of the HI.V-1 LTR promoter essential for viral replication) and accessory genes (vpr, vpu, vif and nef). Preferably, the EMV
gene of H1V-1 can be replaced with the VSVG gene and the residua] HW 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 anuiiotic cells can express some marlcers nomially present on stenz 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(l): 9-12; Wei et al., Cell Transplant., 2003, 12(5): 545-52.
Moreover, evidence may suggest absence or poor expression of HLA-A, HLA-B, HLA-C and HLA-DR
antigens and 0 2 microglobulin on the surface of amniotic cells. Akle et al., Lancet, 1981, 2(8254):
1003-5; Adinolfi et al., Nature, 1982, 295: 28. The HAC according to the invention can express BLA class lb (HLA-G), which may provide for low ixnmune rejection and a long period of survival in a host. Similarly, HAC can undergo differentiation after xeno- or allo-transplantation. Ueta et al., Clin. Exp. Irnmunol., 2002, 129(3): 464-70.

Promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources such as, for example, RNAi 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.
Exenlplary marker genes can include the E. Coli lacZ gene, which encodes P-galactosidase, green fluorescent protein (GFP) or the enhanced green fluorescent protein (EGFP).
In one embodiment, 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. For example, 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. Bemelmans et al., Hum. Gene Ther., 1999, 10: 2987-97;
Ghodsi et al., Hum. Gene Ther., 1998,9: 2331-40; Snyder et al., Nature, 1995, 374: 367-70;
Palella et al., Gene, 1989, 80: 137-44; Bjorklund et al., Brain Res., 2000, 886: 82-98; Kang et al., Hmn.
Cell, 2001, 14: 39-48; Yamada et al., Proc. Natl. Acad. Sci., 1999, 96: 4078;
Tuszynski et al., Exp. Neuro1.,1998,154: 573-82; Saille et al., Neuroscience, 1999, 92: 1455-63;
Haase et al., Ann. Neuro1.,1999, 45: 296-04; Mohajeri et al., Hum. Gene Ther., 1999,10: 1853-66;
Azzouz et al., Hum. Mol. Genet., 2000, 9: 803-811; Adachi et al., Hum. Gene Ther., 2000, 11: 77-89. Preferred exogenous gene elements can comprises glia-derived neurotrophic factor (BDNF) or brain-derived neurotrophic factor (GDNF) genes.
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. In one embodiment, 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.
During adulthood, 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. Several CNS disorders are also associated with a decrease in trophic support. Given that 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, Parldnson'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 uptalce 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 inesencephalic dopaminergic neuron and has been shownto ameliorate motor deficits and reduce brain damage in several aivmal models.
Bjorklund et al., Brain Res., 2000, 886: 82; Gash et al., Nature, 1996, 380:
252; Kordower et al., Science, 2000,290: 767; Zurnet al., Brain Res. Rev., 2001, 36: 222.
One example of an 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. In one embodiment, the exogenous gene elements can be constTUcted or modified through conventional recombinant techniques.
Another aspect of the invention includes a composition comprising the human amniotic cells and at least one pharmaceutically acceptable carrier. For example, HAC
comprising lentiviral vectors with exogenous gene elements and compositions thereof can be transplanted into the body of a subject for gene therapy. Preferably, 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 whicli 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. Moreover, cerebral hemorrhage is a sudden loss of consciousness resulting from the rupture or occlusion of a blood vessel, leading to oxygen laclc 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 limitation, 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. In general, brain tuinors 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 dopaniine (a neurochemical) throughout the brain.

This destruction occurs due to genetic or environmental factors as well as combinations thereof.
In one embodiment, intracerebral grafting of GDNF-transduced HAC (for example, epithelial) into ischemic rats prepared by middle cerebral artery occlusion (MCAo) can significantly ameliorate behavioral dysfunctions and reduce infarct volumes.
Furthermore, neuronal markers and markers of neuronal stem cells can be detected in these transplanted HAC. Similarly, a number of these transplanted HAC survive and migrate to the infarct area.
For example, 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). In the brain, 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. According to the invention, grafting of BDNF-transduced HAC into a Rhesus monkey with incomplete dorsal spinal cord injury can also significantly ameliorate behavioral dysfunction.
As used herein, "optional" or "optionally" can mean that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which it does not.
As described, 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, microcrystalline cellulose, amylum, PVP, agar, pectin, magnesium aluniinate 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 105 to 1010. 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.

The examples herein are provided to illustrate advantages of the present invention that have not been previously described and to further assist a person of ordinary skill in the art with preparing and using the HAC thereof. The examples can include or incorporate any of the variations or embodiments of the invention described above. The embodiments described above may also further each include or incorporate the variations of any or all other embodiunents of the invention. Moreover, biological methods involving conventional techniques are described herein. Such conventional techniques are generally known in the art and are described generally in, for example, Molecular Cloning: A Laboratory Manual, 3rd Edition, vol. 1-3, ed. Saiiibrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001 and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, N.Y., 1992. Similarly, exeniplary methods for the chemical synthesis of nucleic acids are described, without limitation, in Beaucage et al., Tetra.
Letts., 1981, 22: 1859-12 and Matteucci et al., J. Am. Chem. Soc., 1981, 103:
3185. For exan-iple, chemical synthesis of nucleic acids can be performed using coinmercial automated oligonucleotide synthesizers. Immunological methods are also described in, without limitation, Methods of hnmunological Analysis, ed. Masseyeff et al., John Wiley & Sons, N.Y., 1992. Conventional methods of gene transfer and gene therapy can also be adapted for use in accordance with the present invention. Gene Therapy: Principles and Applications, ed.
Blackenstein et al., Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. Robbins et al., Humana Press, 1997; Retro-vectors for Hiunan Gene Therapy, ed. Hodgson et al., Springer Verlag, 1996.

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.
Vectors Metllods of malcing paclcaged retroviral and leiitiviral packaging systems are well laiown to those of ordinaiy slcill in the art. Briefly, lentiviral vectors were produced by transient transfection of 293T cells. 20 g of lentiviral vectors (PWPT, LVTHM
or PWPT-GDNF, Fig. 1), l0,ug of pMDlg/pRRE (or pCMVdR 8.2), 51u.g of pMD2 G and 51tg of pRSV-REV were mixed and adjusted to 250 l with water. 250 l of CaC12 0.5 M were mixed therewith and to the resultirig mixture was added 500 l of HeBS2X (0.28 M
NaCI, 0.05 M
HEPES, 1.5 M Na2HPO4), wllich was still for 30 minutes on a bencll.

The dishes were placed in a 37 C humidified incubator with a 5 % CO2 atmosphere.
The medium was aspirated. 14 hours later, 10 ml of fiesh DMEM-10 % FBS (PAA
Austria) prewarmed to 37 C was gen.tly added, followed by incubation for 28 llours. The virus was collected and cleared via centrifiigation at 1500 ipm for 15 minutes and filtered through a 0.45 m filter. Ultracentrifi.lgation occurred for 90 ininutes at 80,000 g and 4 C. An aliquot of the supeniatant was resuspended as pellets with 1 ml PBS and stored at -80 C. Titration of the concentrated supenlatants was perfonned by serial dilutions of vector stocks on 1x 105 Hela cells, followed by fluorescence-activated cytometric, Beckton Dickinson hrununocytometry Systems, analysis. According to the fonnula, 1 x 105 Hela cell x (%) EGFP positive cells x 1000/ l virus, titers of lentiviral vectors were calculated among 0.1-1 x 10' TU/ml.

HAC infected with lentiviral vectors The viral vector was added to cultured amniotic cells on the following day for 2 days in the presence of 1-10 g/rnl polybrene (Sigma-Aldricli). After washing with PBS, HAC
continued to be cultured in a cell culture medium such that exogenous gene elements could be introduced into HAC.
EXAlVIl'LE II
Lentiviral vector infection Studies and use of lluinan amnion were approved by both patient and etlucs conunittee review. For example, an ainnion meinbrane 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 mediunl and cut witli 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 gg/ml, penicillin 100 U/ml and glutamine 0.3 mg/ml, followed by incubation under a humidified atiuosphere of 5 % CO2 in air at 37 C. HAC were plated on a 6-well plate (2 x 105 cells/well, Costar) and polybrene (Sigma-Aldrich) was added to the wells for a final concentration of 8 g/ml.
Lentiviral vectors (PWPT) were added (MOI = 100), after 48 hours of incubation. DOTAP
(Boehringer Mannheim) was also used to transfect HAC.
Transduced HAC was cultured for 1 weelc. EGFP expression was visualized by fluorescent micropliotograph and analyzed by fluorescence-activated cell sorting (FACS).
Genoinic DNA was isolated as described by, for example, Promega and total RNA
isolation and reverse transcription were performed (Promega). To measure the relative expression of EGFP, a semi-quantitative PCR for EGFP and (3-actin (internal reference) analysis was performed with PCR amplification for 28 cycles. Primer sequences were EGFP
(upstream 5'-cgagctggacggcgacgtaaac-3' and downstream 5'-gcgcttctcgttggggtctttg-3') and (3-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 RT-PCR products was 597 bp. The expression difference was normalized by the respective (3-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.

Results EGFP can be observed with fluorescence microscopy in the 4th day after transduction.
As a control, HAC was transduced with DOTAP and these cells were scored by fluorescence-activated cell sorting (FACS) in the 7th day. 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 1o 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). To evaluate integration of EGFP into HAC, genomic DNA and 19, mRNA was extracted in the 7tli day after transduction and subjected to PCR and RT-PCR
amplification. The results showed that EGFP was inserted into the genome of HAC with stable expression at the mRNA level, (Fig. 2C). Lentiviral vectors did not affect the percent of GO-Gl stage cells after transfection, (Fig. 2D), demonstrating that lentiviral vector modified HAC were steady gene transniitters. As such, the 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.

EXAMPLE III
Lentiviral. vectors-mediated siGFP suppression in HAC
HAC were plated on a 6-well plate (2 x 105 cells/well, Costar) aild polybrene (Sigma -Aldrich) was added to the wells. With a final concentration of 8 g/ml, lentiviral vectors (pLVTHM and pLVTHMsiGFP) were added (MOI =100). EGFP expression was visualized by fluorescent microphotograph. and analyzed by fluorescence-activated cell sorting (FACS).
To measure the relative expression of EGFP, a semi-quantitative PCR for EGFP
and J3-actin (internal reference) analysis was performed with PCR amplification for 28 cycles. Primer sequences were EGFP (upstream 5'-cgagctggacggcgacgtaaac-3' and downstream 5'-gcgcttctcgttggggtctttg-3') and 0-actin (upstream 5'-aacgagcggttccgatgccctgag-3' and downstream 5 ' tgtcgccttcaccgttcca.gtt 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 aud 72 C
for 10 minutes. The expected length of the EGFP PCR products was 597 bp. The expression difference was normalized by the respective (3-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.
Results RNA interference was the best gene-silencing pathway of RNIA mediated gene regulation in post-transcription. Expression of siRNAs delivered by lentiviral 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. In 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. In contrast, HAC-siGFP cells cotransduced with the constitutively active PLVTHM and PLVTHMsiGFP exhibited a strong down regulation of EGFP, (Fig. 3C). FACS results demonstrated that PLVTHMsiGFP
silenced EGFP expression to less than 15 % in HAC-siGFP cells, (Fig. 3B).
Based on RT-PCR results, it was found that inhibition was posed in the mRNA level, (Fig.
3D).
EXAMPLE IV
Lentiviral vectors based Cre-loxP systein delete EGFP transduced into HAC
HAC were plated on a 6-well plate (2 x 145 cells/well, Costar) and polybrene (Sigma-Aldrich) was added to the wells. With a final concentration of 8 g/ml, lentiviral vectors (PWPT
and PWPT-Cre) were added (MOI = 100). Transduced HAC were cultured for 1 weelc. 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. To measure the relative expression of EGFP, a semi-quantitative PCR for EGFP and 0-actin (internal reference) analysis was performed with PCR amplification for 28 cycles. Primer sequences were EGFP (upstream 5'-cgagctggacggcgacgtaaaa-3' and downstream 5'-gcgcttctcgttggggtctttg-3') and P-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 (3-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. Mol. 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.
Results FACS results demonstrated that EGFP was silenced to less than 10 %, (Fig. 4A, and 4C). Moreover, it was also found that this deletion was posed in the genomic DNA, (Fig.4D). As such, the specific gene with PWPT-Cre in HAC can be lrnoclced out.

EXAMPLE V
Lentiviral vector based doxycycline-inducible systems regulate EGFP expression HAC were plated on a 6-well plate (2 x 105 cells/well, Costar) and polybrene (Sigma-Aldricli) was added to the wells. With a final concentration of 8 g/ml, lentiviral vectors (pLVTH1VI and lenti-tTRKRAB) were added (MOI = 100). After 48 hours of incubation, doxycycline (final concentration of 5gg /ml, Sigma) was added to PLVTHM and the lenti-tTRKRAB cotransduced cells.

Results During the absence of doxycycline, the tTR-KRAB protein binds specifically to tetO, providing a means for suppression of the activity of the nearby promoter (up to 31cb from its DNA-binding site). Conversely, in the presence of doxycycline, 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). The tTRKRAB
based on lentiviral vectors suppressed the expression of EGFP in the absence of doxycycline, (Fig. 5B). In contrast, addition of doxycycline to the dually transduced cells resulted in EGFP (re)expression, (Fig. 5C).

EXAMPLE VI
Biosafety detection To detect the intergrated vector in HAC, the cells were transduced with PWPT
for 3 days and washed intensively. Thereafter, the medium was collected and filtered through a 0.45 gm filter. Hela cells with the medium were cultured for 3 days and genomic DNA of the Hela 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'-gcgcttctcgttggggtctttg-3') and 0-actin (upstream 5'-aacgagcggttccgatgccctgag-3' and downstream 5'-tgtcgccttcaccgttccagtt 3') as .22-well as primers homologous to the HIV-1 gag gene (upstream 5'-gagtatctgatcatactgtcctac-3' and downstream 5'-ggaactactagtacccttcaggaa-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 minutes. The expected length of EGFP and gag products was 597 bp and 912 bp, 5 respectively. The expression difference was normalized by the respective (3-actins (590 bp).
Ten microliters from each RT-PCR product were loaded on a 1.5 % agarose gel containing 0.5 ggJml of ethidium bromide and separated by electrophoresis.

Results 10 It was fou.nd that EGFP integrated well into the HAC and EGFP did not exist in the genome of the Hela cells, (Fig. 6A). GAG, a structure protein in lentiviruses, which can be necessary for virus formation, was not detected in the EGFP expressed HAC or Hela cells, (Fig. 6B). These results demonstrated that lentiviral infected HAC can have a checlc-up for biosafety before transplantation.
EXAMPLE VII
HAC iuifected and transplanted into cerebral ischemic rats Lenti-GDNF (MOI = 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-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. 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 %. Using a stereotaxic frame (Narishige) and a 26- gauge Hamilton syringe, 8 x 105 GDNF modified HAC (HAC-GDNF) in 5 l of PBS were injected into the right dorsolateral striatum of MCAo rats. Markgraf et al., Brain Res., 1992, 575(2):
238-46. Proximally, the injection was 4 mm beneath the skull surface, 1 mm posterior and 3 mm lateral to the bregma over a 10-minute period, which approximated the ischemic boundary zone. Anterior-posterior (AP) = -1 mm, medial-lateral (ML) = 3 mm and dorsal-ventral (DV) = 4 mm from the bregma.

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 inm 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. Iruage analysis software AutoCAD (AutoDesk) was used for au estimation of the infracted volume. The results were expressed as a percentage of the hemisphere.

Results In all the groups, the infarct volume decreased from day 2 to day 16. Two days after MCAo, there were no significant differences among the two groups. 16 days after MCAo, there was a significant reduction in the percentage of infarct volume in the HAC-GDNF
group compared to rats in the control PBS group, (Fig. 7).

Immunohistochemistry detection For GDNF and MAP2 staining 16 days after transplantation, 3 rats of each group were killed and examined by immunohistochemistry. The brains of the rats were fixed with 4 %
paraformaldehyde fixative for 2 days. After which, 30 gm of frozen sections (near the injection tract) were cut with a cryostat at -20 C and subjected to immunohistochemistry.
The sections were rinsed 3 tiunes in PBS (pH 7.4). Endogenous peroxidase activity was quenched with H20Z (0.3 %) for 30 minutes. After blocking with 10 % normal goat serum or horse serum for one night in 4 C, 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 bybiotin 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). Lastly, the slide was colorized with diaminobenzidine (DAB). The immunohistochemical studies were repeated at least three times.
Results GDNF engineered HAC (HAC-GDNF) were injected into the lateral striatum and cortex of MCAo rats and the grafts were detected by immunohistochemistry in the brains of the subjects. There were no positive signals for'GDNF in the cortical area of the normal nontransplanted rat (not shown). GDNF-positive cells were detected in the injection tract of the ischemic rats brains including in the cortex and striatum. A number of GDNF positive cells were also found in HAC transplanted MCAo rats, (Fig. 8). 16 days after transplantation, MAP2 positive cells were found in the injection tracts, (Fig. 9), demonstrating that the HAC
have the potential to differentiate into neurons.

Neurological examination The neurological findings were scored on a modified scoring system that was developed by Longa et al. For example, a score of 0 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 :LSE with a significance level of P < 0.05.
No neurological deficits were observed before MCAo. Rats in the strolce groups (stroke with PBS and stroke with GDNF transduced HAC) were examined at 4 time points after cell transplantation, up to 16 days.

Results The neurological findings scored on a six-point scale were demonstrated and data indicated significant differences in the two groups, (Fig. 10). Follow-up comparison analyses revealed that there was significant difference between HAC-GDNF groups and the PBS
group (p < 0.05) in the fourth day. These results indicate that HAC-GDNF
significantly reduced the severity of neurological deficits for rats especially in the early stage.

Moving test (beam-walking test) 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 wallc when a wall is placed next to the beam). Scoring was from 0 to 6. In particular, for 0, 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 wallcing, for 3, the rat can traverse the beam, but the affected hindlimb 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. Treatinent was examined and analyzed statistically by mean +SE with a significance level ofP < 0.05.

Results Performance of the beam-walking test sllowed differences among the tliree animal groups at four time points from day 4 to day 16, after traiisplantation.
Statistically significant improvement effects were detected in HAC-GDNF transplantation over time, (Fig.
11).
Furthermore, the performances of the HAC-GDNF groups were significantly better than the control group in the early stage. Moreover, impaired coordination function in the non-treated ischemic rats (control group) did not recover.
These results suggest that 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.
These results may be caused by the neurotrophic factors or anti-inflammation factors secreted by HAC as well as differentiation to neuronal cells.
EY_AMPLE VIII
Preparation of cell injection As described herein, HAC-GDNF were prepared and suspended in PBS.
EXAMPLE IX
Grafting HAC infected with PLVTHM-BDNF in rats with Parkinson's disease PLVTHM BDNF (MOI = 100) 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 108.
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. Three weelcs later, the subjects were tested with apomorphine, which causes aberrant rotation behavior in recipients with a successful biochemical lesion of the nigral-striatal tract. In the cell transplantation group, 8 x 105 BDNF
modified HAC in 5 l of PBS were injected into the striatum of the 6-OHDA
model of the PD rat (AP = -5.0 mm, ML = 2.5 mm, DV = -6.5 mm). In the control group, 5 l of PBS
was injected into the striatum of the 6-OHDA model of the PD rat (AP = -5.0 mm, ML =
2.5 mm, DV =--6.5 mm). Thereafter, at 2, 4 and 8 weelcs later, apomorphine-induced rotations were observed.

Results In the PD rats, HAC infected with PLVTHM-BDNF led to significant reductions in apomorphine-induced rotations, (Table 1).

Table 1 Comparison of the total apomorphine-rotations in the two groups Time (week) Cell transplantation group Control group 0 260.2 :0.6 233.3 12.2 2 297.6 5.4 96.5 8.8 4 243.7 2.8 99.4 5.9 8 233.5 8.6 52.9 10.2 EXAMPLE X
Grafting HAC infected with PLVTHM BDNF in a Rhesus monkey with incomplete dorsal spinal cord injury PLVTHM-BDNF (MOI = 80) was added to DMEIVI-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 IUPBS and mechanically dissociated into a single cell suspension. The cell concentration was adjusted to 1-2 X 108. 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. In the cell transplantation group, 1 x 10' BDNF modified HAC were injected into the injured position of 2 monkeys.
In the control group, PBS was injected into the injured position of one monlcey. Two months later, a BBB locomotor rating scale was evaluated. Basso et al., J. Neurotrauma, 1995,12(1): 1-21.
Results The BBB score of the cell transplantation group was 9.7. The BBB score of the control group was 5.2. As such, the HAC infected with PLVTHM BDNF were shown to improve hindlimb motor function of the subject.

While the present invention has been described herein in conjunction with a preferred embodiment, a person with ordinary skill in the art, after reading the foregoing, can effect changes, substitutions of equivalents and other types of alterations to that set forth herein.
Each embodiment described above can also have included or incorporated therewith such variations as disclosed in regard to any or all of the other embodiments.
Thus, it is intended that protection granted by Letter Patent hereon be limited in breadth and scope only by definitions contained in the appended claims and any equivalents thereof.

Claims (18)

1. A population of human amniotic cells comprising lentiviral vectors, wherein the lentiviral vector comprises an exogenous gene element capable of being expressed by the human amniotic cells.
2. The population of human amniotic cells of claim 1, wherein the exogenous gene element encodes a 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.
3. The population of human amniotic cells of claim 1, wherein the exogenous gene element encodes a glia-derived neurotrophic factor.
4. The population of human amniotic cells of claim 1, wherein the exogenous gene element encodes a brain-derived neurotrophic factor.
5. The population of human amniotic cells of claim 1, 2, 3 or 4, wherein the lentiviral vector comprises at least one controlling transcription fragment of an RNAi-inducible, Cre-loxP or doxycycline-inducible system.
6. A composition comprising the population of human amniotic cells of claim 1, 2, 3, 4 or 5 and a pharmaceutically acceptable carrier.
7. A human amniotic cell comprising a lentiviral vector, wherein the lentiviral vector comprises an exogenous gene element capable of being expressed by the cell.
8. The human amniotic cell of claim 7, wherein the exogenous gene element encodes a 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.
9. A method of transducing a population of human amniotic cells with a lentiviral vector comprising incubating at least one lentiviral vector with the population of human amniotic cells.
10. A method of treating central nervous system disease in a subject using a population of human amniotic cells comprising a lentiviral vector with an exogenous gene element.
11. The method of claim 10, wherein the central nervous system disease comprises cerebral ischemia, cerebral hemorrhage, central nervous system trauma, hereditary diseases of the nervous system, neurodegenerative diseases or neoplasms of the central nervous system.
12. The method of claim 10, wherein the central nervous system disease comprises cerebral ischemia, central nervous system trauma or neurodegenerative diseases.
13. The method of claim 10, wherein the central nervous system disease is cerebral ischemia.
14. The method of claim 10, wherein the central nervous system disease comprises spinal cord trauma.
15. The method of claim 10, wherein the central nervous system disease is Parkinson's disease.
16. A method for treating central nervous system diseases comprising administering to a subject in need thereof, an effective amount of human amniotic cells comprising a lentiviral vector, wherein the lentiviral vector comprises an exogenous gene element capable of being expressed by the cells.
17. The method of claim 16, wherein the exogenous gene element encodes a 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.
18. The method of claim 16, wherein the central nervous system disease comprises cerebral ischemia, cerebral hemorrhage, central nervous system trauma, hereditary diseases of the nervous system, neurodegenerative diseases or neoplasms of the central nervous system.
CA002620946A 2005-06-24 2006-06-23 Amniotic cells and methods for use thereof Abandoned CA2620946A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CNA2005100271288A CN1884495A (en) 2005-06-24 2005-06-24 Human amnion cell capable of expressing extraneous gene and its preparation method and uses
CN200510027128.8 2005-06-24
PCT/CN2006/001437 WO2006136114A1 (en) 2005-06-24 2006-06-23 Amniotic cells and methods for use thereof

Publications (1)

Publication Number Publication Date
CA2620946A1 true CA2620946A1 (en) 2006-12-28

Family

ID=37570119

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002620946A Abandoned CA2620946A1 (en) 2005-06-24 2006-06-23 Amniotic cells and methods for use thereof

Country Status (7)

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

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0514567D0 (en) * 2005-07-15 2005-08-24 Univ Nottingham Surgical membrane
EP2067858A1 (en) * 2007-12-07 2009-06-10 Universidad de Sevilla Animal models for neurodegenerative diseases
CN101721430B (en) * 2008-10-22 2013-01-23 中美赛傲(上海)生物技术有限公司 Application of amniotic epithelial cells in pharmacy
CN103705979B (en) * 2013-09-18 2016-10-05 中国人民解放军海军总医院 For functional organization's engineering material of CO2 laser weld and preparation thereof and purposes

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3806189B2 (en) * 1995-07-27 2006-08-09 株式会社エスアールエル Methods for gene transfer into human amniotic cells and methods for preparing cells for gene therapy
JP4217037B2 (en) * 2001-08-10 2009-01-28 宣男 櫻川 Human neural stem cells derived from human amnion mesenchymal cells
AU2002353231B2 (en) * 2001-12-21 2008-10-16 Oxford Biomedica (Uk) Limited Method for producing a transgenic organism using a lentiviral expression vector such as EIAV
WO2004000164A2 (en) * 2002-06-24 2003-12-31 Amniotech, Inc. Amniotic membrane mediated delivery of bioactive molecules

Also Published As

Publication number Publication date
CN1884495A (en) 2006-12-27
EP1893750A1 (en) 2008-03-05
KR20080025173A (en) 2008-03-19
JP2008543319A (en) 2008-12-04
AU2006261485A1 (en) 2006-12-28
WO2006136114A1 (en) 2006-12-28
EP1893750A4 (en) 2009-05-13

Similar Documents

Publication Publication Date Title
EP2520302B1 (en) Stromal cell-derived factor-1 mediates stem cell homing and tissue regeneration in ischemic cardiomyopathy
US20040161412A1 (en) Cell-based VEGF delivery
CN111225682A (en) Use of syncytin for targeted delivery of drugs and genes to lung tissue
US20150322410A1 (en) Hypoxia regulated conditionally silenced aav expressing angiogenic inducers
US20210260168A1 (en) Compositions and methods of fas inhibition
CA2620946A1 (en) Amniotic cells and methods for use thereof
Liu et al. Republished review: Gene therapy for ocular diseases
US20100184838A1 (en) Compositions and methods for retinal transduction and photoreceptor specific transgene expression
JP2024519218A (en) Method for producing mature corneal endothelial cells
US20220127575A1 (en) Genetically modified hematopoietic stem and progenitor cells (hspcs) and mesenchymal cells as a platform to reduce or prevent metastasis, treat autoimmune and inflammatory disorders, and rebalance the immune milieu and dysregulated niches
US10744162B2 (en) Genetically modified muscle cells which express neurotrophic factors
EP3795170B1 (en) Pharmaceutical composition comprising ccn5 as active ingredient for preventing or treating retinal diseases
WO2022244851A1 (en) Preventive agent and/or therapeutic agent for bedsores
WO2002050263A2 (en) Methods_for treating tissue damage by bone-marrow derived stem cells
JP2002514409A (en) Genetically modified fibroblasts and uses thereof
KR101663811B1 (en) Pharmaceutical compositions for preventing or treating brain diseases
by Chimeric et al. GENE THERAPY OR THE NERVOUS SYSTEM I
JP2014039516A (en) Proliferation control type virus vector comprising synoviolin promoter
Steinmetz et al. Cellular and Gene Therapy Approaches to Spinal Cord Injury
AU2008200822A1 (en) Stromal cell-derived factor-1 mediates stem cell homing and tissue regeneration in ischemic cardiomyopathy

Legal Events

Date Code Title Description
FZDE Discontinued