KR20080025173A - Amniotic cells and methods for use thereof - Google Patents

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. ® KIPO & WIPO 2008

Description

Amniotic cells and methods for use

The present invention relates to the fields of molecular biology, gene therapy, immunology and virology. More specifically, the present invention relates to human amniotic cells comprising lentiviral vectors having exogenous gene elements, methods for producing such human amniotic cells and methods for treating diseases.

Humans are for example cerebral ischemia, cerebral vascular disease, central nervous system damage, hereditary diseases of the nervous system, degenerative diseases of the brain nervous system (e.g. Parkinson's disease, Huntington's disease and Alzheimer's disease) and Suffer from central nervous system diseases such as tumors of the brain's nervous system. To date, there are few effective treatments or drugs for central nervous system disease. In general, the rate of death or disability caused by central nervous system disease is very high. For example, 16,400,000 people worldwide suffer from brain strokes, brain injuries and spinal cord injuries every year. In addition, 4,100,000 of these people die each year from the disease.

Recently, gene therapy has developed as a method with potential efficacy for the treatment of many neurological diseases previously considered intractable in the traditional manner. In general, there are three strategies for gene therapy to treat central nervous system disease. The first is to reduce the activity of mutant recessive gene proteins with antisense technology and RNAi technology. For example, mutant recessive genes of central nervous system disease express proteins, and gene therapy can be used to down-regulate the expression of the mutated mRNAs via antisense or RNAi. Another strategy is to compensate for loss-function enzymes or proteins in the brain by delivering genes. Finally, neurons can be protected by the delivery of proteins such as growth factors, antioxidants, HSPs or anti-apoptotic molecules. Natsume et al., Exp. Neurol., 2001, 169: 231-33; Berry et al., Curr. Opin. Mo L Ther., 2001, 3: 338-49.

Currently five types of viral vectors are used for gene therapy. (1) retroviral vectors that infect only proliferating cells, and (2) adenoviral (Ad) vectors (Ad vectors that cannot stably express without insertion into the chromosome of a host cell may also produce immunogenicity. ), (3) adeno-associated viral (AAV) vectors that cannot deliver large exogenous fragments (less than 5Kb), or have high titers, (4) infect only neurons Are herpes simplex viruses (HSV) and (5) lentiviral vectors. Currently, there are many problems with the efficacy, stability, controllability and safety of using genetically engineered viral vectors directly for gene therapy in vivo. These problems include, for example, the stability and specificity of viral vectors in vivo. In addition, the use of embryonic stem cells or somatic stem cells for gene therapy in ex vivo is of concern with regard to safety, immune rejection, cell origin 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 Palma et al., Nat. Med, 2003, 9 (6): 789-95; Imren et al., J. Clin. Invest, 2004: 953-62.

The present invention, in one aspect, relates to a population of human amniotic cells (HAC) comprising a lentiviral vector having at least one exogenous gene element. Preferably, the exogenous gene element of said vector can be expressed by said cell. In 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 transforming human amnion cells. Another object of the present invention is to provide a method for treating central nervous system disease using human amnion cells comprising a lentiviral vector having at least one exogenous gene element. For example, the methods of the present invention comprise administering to a patient human amnion cells comprising a lentiviral vector having at least one exogenous gene element.

The present invention also provides transfected human amnion cells that may be useful for gene therapy. Preferably, the exogenous gene element may be highly expressed in human amnion cells comprising a lentiviral vector. These cells include central nervous system diseases such as cerebral ischemia, cerebrovascular disease, central nervous system damage, hereditary diseases of the nervous system, brain neurodegenerative diseases (e.g., Parkinson's disease, Huntington's disease and Alzheimer's disease), cerebral nervous system tumors, and combinations of these diseases. Can effectively cure The cells can also be used to treat any of the diseases disclosed herein. In one embodiment, human amniotic cells according to the present invention can minimize or eliminate rejection or local inflammation. Prior to transduction, the human amnion cells may be derived from a donor, tissue culture, or individual (eg, a patient), but are not limited to these.

In one embodiment human amniotic cells transformed with a lentiviral vector are also used for cerebral ischemia, cerebrovascular disease, central nervous system damage, inherited diseases of the nervous system, brain neurodegenerative diseases (eg, Parkinson's disease, Huntington's disease and Alzheimer's disease). It may be used in the treatment or prophylaxis of diseases including cerebral nervous system tumor diseases and combinations of these diseases, but is not limited to these examples. Human amnion cells transformed with the lentiviral vectors of the invention can also be used for the treatment or prevention of any disease state or lady disclosed herein. In general, "prophylactic" or "prophylaxis" relates to a reduction in the likelihood of a patient developing with or progressing to a disorder such as Alzheimer's disease (AD). For example, human amnion cells transformed with the lentiviral vectors of the present invention can be used prophylactically as a measure designed to maintain patient health and prevent the spread and maturation of the disease. The present invention also provides a method of administering to a patient an effective amount of human amnion cells transformed with a lentiviral vector for the treatment or prevention of a disease, such as, for example, CNS disease.

Human amnion cells transformed with the lentiviral vectors of the invention can also be administered to a patient in combination with other traditional therapeutic measures or agents that may be useful, for example, for the treatment or prevention of CNS diseases. In one embodiment there is provided a method of administering an effective amount of human amnion cells transformed with a lentiviral vector of the invention to a patient suffering from or believed to be at risk of suffering from a disease. The method also includes administering human amniotic cells transformed with the lentiviral vectors of the invention in a sequential or in combination with a traditional therapeutic agent in an amount that may be potentially potent in the treatment or prevention of CNS disease.

Preferably, human amnion cells transformed with the lentiviral vectors of the present invention may be administered to the patient in an appropriate amount or dosage to treat the CNS. In general, dosages or compositions comprising human amnion cells transformed with the lentiviral vectors of the invention vary depending on considerations. For example, such considerations include age, condition, sex, degree of disease, contraindications, and accompanying treatment. Exemplary dosages or compositions may also be adjusted or modified by one skilled in the art based on the above considerations. Administration of human amniotic cells transformed with the lentiviral vectors of the present invention to an individual is local or systemic, intravenous, intraarterial, intradural (via spinal fluid), and the like. Can be achieved by Administration can also be intradermal or intracavitary, depending on the part of the body being treated. A "subject" is, for example, a mammal, such as a human, and preferably a human suspected of having one or more CNS diseases. The terms "subject" and "patient" are used interchangeably herein.

Human amniotic cells transformed with the lentiviral vectors of the invention can also be administered in the form of compositions, such as injectable compositions, but also, for example, topical, oral, rectal, parenteral Formulated in well-known drug delivery systems such as intravenous, intramuscular or subcutaneous, intracisternal, vaginal, intraperitoneal, topical (powder, ointment or drops) or transplantation, buccal or nasal spray Can be. As noted above, administration of human amniotic cells transformed with lentiviral vectors or compositions thereof can be local or systemic and can be achieved intravenously, intraarterally, intradurally (via the spinal fluid) or through transplantation. Administering human amniotic cells transformed with a lentiviral vector to an individual can be by a general or topical route of administration. For example, human amnion cells transformed with a lentiviral vector or a composition thereof can be administered to the patient for delivery throughout the body. In addition, human amnion cells or compositions thereof transformed with the lentiviral vectors may be administered to specific organs or tissues.

Exemplary compositions for administration may comprise a pharmaceutically acceptable carrier for human amnion cells transformed with one or more lentiviral vectors of the invention. Pharmaceutically acceptable carriers include, for example, aqueous solutions and carriers such as nontoxic 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 human amnion cells transformed with lentiviral vectors include propylene glycol, polyethylene glycol, methoxypolyethylene glycol and plant oils or Non-aqueous solvents such as injectable organic esters such as ethylene oleate. In one embodiment human amnion cells transformed with a lentiviral vector may be conjugated with at least one pharmaceutically acceptable carrier. Aqueous carriers may include, but are not limited to, water, alcohol / aqueous solutions, physiological saline and parenteral vehicles such as sodium chloride or Ringer's dextrose. Intravenous carriers for administration of human amniotic cells transformed with the lentiviral vectors of the invention may include, for example, fluid and nutrient replenisher. In addition, the pH and the exact concentration of the various components of the composition comprising human amnion cells transformed with the lentiviral vector can be adjusted by 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 human amnion cells transformed with a lentiviral vector. The present invention also provides a method for the treatment or prevention of CNS disease comprising administering to a patient in need of a human amniotic cell transformed with an effective amount of a lentiviral vector. For example, the method may include providing a patient with or believed to have a CNS disease. The method also includes administering to the patient an effective amount of human amnion cells transformed with the lentiviral vector of the invention. Human amnion cells transformed with the lentiviral vectors 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 achieve the desired effect. One example of an effective amount includes an amount or dosage that can be used to mitigate or minimize the effects of CNS disease. Another example of an effective amount includes an amount or dosage that produces an acceptable level of toxicity and bioavailability for therapeutic use, including the treatment or prevention of CNS disease. Another example of an effective amount includes an amount or dosage that can minimize or interfere with neuronal degeneration. If desired, human amnion cells or compositions thereof transformed with the lentiviral vectors of the invention may be pH modifiers (e.g. acids, bases, buffers), stabilizers (e.g. ascorbic acids) or isotizing agents. Additives such as (eg, sodium chloride).

A person of ordinary skill in the art can administer a human amniotic cell or composition thereof transformed with a lentiviral vector by simply administering the cell or composition thereof to the individual while increasing the amount over a period until the effect of the disease is reduced. Effective amounts can be easily determined. Effective determination of the effective amount for a particular subject is well known to those of ordinary skill in the art. The present invention also contemplates that human amnion cells can be used for gene therapy in ex vivo or in vivo. Preferably, for in vivo genetic difference treatment of CNS disease, an effective amount of retrovirus, AAV, lentiviral, pseudotyped lentiviral or Ad vector for transformation of human amnion cells transformed with lentiviral vector Can be administered to the subject. These vectors can be administered in a composition comprising a pharmaceutically acceptable carrier. In addition, these vectors can be administered by topical, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), control, intravaginal, intraperitoneal, topical (powder, ointment or drops) or implantation, buccal or nasal sprays. Can be. As noted above, administration can also be accomplished locally or systemically and intravenously, intraarterally, intradurally (through the spinal fluid) or through transplantation.

In addition to using a promoter for expression in human amnion cells, enhancer sequences can be used to increase expression levels. Armelor et al, Proc. Natl. Acad. ScL, 1973, 70: 2702. Exemplary growth factors encoded by exogenous gene elements are neuronal growth factor, neurotrophic factor, and brain derived growth factor (BDNF). ), Neurotrophin (NT) -3, NT-4 and ciliary neuronal trophic factor (CNTF).

In addition, the present invention is a nerve growth factor, brain-derived neurotrophic factor, hypoxanthine guanine phosphoribosyltransferase, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L-amino acid dicarboxyl Analogs, homologs, derivatives and variants of ase, bcl-2 or tetrahydrobiopterin synthase are contemplated. Similarly, the present invention relates to nerve growth factor, brain-derived neurotrophic factor, hypoxanthine guanine phosphoribosyltransferase, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L-amino acid dica Analogs, homologues, derivatives and variants of exogenous genetic elements encoding reboxylase, bcl-2 or tetrahydrobiopterin synthase are contemplated. Exogenous elements may also be tyrosine hydroxylase, GTP-cyclohydrolase I, aromatic amino acid dopa decarboxylase, vesicular monoamine transporter 2 (VMAT2) or any suitable protein. Or to encode an antibody.

Moreover, the present invention contemplates transformation of human amniotic cells via one or more retroviruses, AAV, lentiviral, pseudolentiviral, Ad vectors and combinations thereof, but is not limited to these examples. For example, these vectors can be used sequentially or in combination to transform human amnion cells. The vector also contains one or more exogenous elements.

In one embodiment a population of human amniotic cells comprising one or more lentiviral vectors is provided. For example, the lentiviral vector may comprise at least one exogenous gene element expressed by the cell. Exemplary exogenous gene elements are neuronal growth factor, brain-derived neurotrophic factor, hypoxanthine guanine phosphoribosyltransferase, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase, aromatic L-amino acid Decarboxylase, bcl-2 or tetrahydrobiopterin synthase. Preferably, the lentiviral vector may comprise a regulatory transcription fragment of at least one of an RNAi-induced, Cre-loxP or doxycycline-induced system. The invention also provides compositions comprising one or more lentiviral vectors comprising at least one exogenous gene.

Also provided are methods of transforming a population of human amniotic cells with one or more lentiviral vectors. Preferably, the method comprises culturing human amnion cells. For example, the amniotic membrane can be separated from the placenta obtained from a donor. The placenta can be scraped extensively to remove underlying tissues. The amnion may optionally be treated with at least one enzyme. In one embodiment the isolated cells can be cultured in cell culture medium. The method also includes culturing at least one vector and the population of human amniotic cells.

In one embodiment a method of treating a central nervous system disease in an individual is provided. Preferably, the method relates to the use of a population of human amniotic cells comprising a lentiviral vector with exogenous genetic elements. 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), degenerative diseases of the nervous system, spinal cord trauma and neoplasms of the central nervous system Include.

Indicates the introduction of genetic material into cells such as human amniotic cells using a transduction or transfection vector such as retrovirus, AAV, lentiviral, pseudolentiviral or Ad vector. Commonly used for betting. Other types of vectors can be used for such transformation or transfection of human amnion cells. These vectors include plasmids or viral vectors. Retroviral vectors such as those based on the Moloney murine leukemia virus (MoMLV) can be used. In addition, other rat retroviral vectors that can be used include those based on rat embryonic stem cell virus (MESV) and rat somatic virus.

Also, lentiviral vectors, a subclass of retroviral vectors, can be used for high efficiency transformation, transform into non-dividing cells, and increase the likelihood of cells becoming pluripotent. Let's do it. In addition, other groups of retroviruses, such as spumaviruses, can efficiently transform non-dividing cells. Additional types of viral vectors that can be used in the present invention include the form of adenovirus vectors, AAV, SV40 based or hybrid vectors. Various expression vectors can be used to deliver genetic elements, such as endogenous or exogenous gene elements encoding bioactive substances, into human amnion cells. The expression vectors may be viral vectors such as modified or recombinant retroviruses, adenoviruses, lentiviruses, pseudolentivials and AAV. In addition, the expression vectors may be non-viral such as physical methods, including electroporation, ultrasound, chemical, liposome mediated, activated-dendrimer-mediated techniques, and phosphate-calcium techniques. May be transfected into the cells via a) method, but is not limited to these examples.

Examples of promoters useful in the present invention include constitutive or inducible promoters. The promoter may include CMV, SV40, retroviral LTR, tetracycline inducible, EF, inflammatory inducible, TNF-α and IL-1 promoters, but is not limited to these examples. In addition to human amnion cells, the present invention may include various other components and means for facilitating delivery of such cells to a subject. For example, human amnion cells can be provided in a medium in which the cells are preserved or maintained. Examples of such media include PBS, DMEM and any suitable type of cell culture medium. In one embodiment the invention contemplates a buffer such as HEPES, PBS or citric acid based buffer, but is not limited to these examples. In addition, the present invention may include dyes, packaging, kits, instructions for using transformed human amnion cells, DMSO and glycerol for cryopreservation. Transformed human amnion cells of the invention can be used for therapy, diagnosis, cosmetics or any other application, but are not limited to these examples.

In one embodiment the exogenous elements may include, but are not limited to, growth factors, antimicrobial proteins, anti-inflammatory proteins or protease inhibitors. Examples of growth factors may include PDGF, FGF-2, EGF, KGF-2, GM-CSF, TGF-b, IGF-I and HGH. In addition, examples of antimicrobial proteins include proteins that increase bactericidal / permeability, defensin, collectin, granulysin, protegrin-1, SMAP-29 , Lactoferrin and calgranulin C. Preferably, examples of anti-inflammatory proteins may include interleukin-1 receptor antagonists, interleukin-10, soluble TNF receptors and soluble CTLA4. Protease inhibitors may also include, for example, TIMP-1, TIMP-2, TIMP-3, TIMP-4, PAI-1, PAI-2 and ecotin.

The present invention also contemplates that each of said vectors for transfection of human amnion cells may comprise at least one promoter. The exogenous genetic element of the vector may also comprise marker sequences. In one embodiment the cells that can be transformed by the vector include mammalian tissue, epithelial cells, alveolar cells, bone marrow, myocardium, connective tissue, ventricular cells, epithelial tissue, epithelium, epidermis, esophagus, fibroblasts, glial cells, liver cells , Keratin cells, white blood cells, lymphocytes, macrophages, mammary glands, melanocytes, monocytes, myocytes, neurons, osteoblasts, osteogenic cells, pituitary cells, plasma cells, skeletal muscle, smooth muscle, synovial cells, navel tissues, human amnion Cells derived from or combining the cells or combinations thereof, but are not limited to these examples. In addition, the exogenous genetic element of the vector may comprise a sequence encoding a detectable marker, such as, for example, green fluorescent protein (GFP).

In one embodiment, human amnion cells transformed with a lentiviral vector can be used to prepare a graft composition. The implant composition may comprise cells transfected with a vector comprising endogenous or exogenous genetic elements. For example, the implant composition may also comprise a biocompatible matrix. The human amniotic cells of the implant composition can be obtained from an individual and transfected in vitro before being reinjected into the individual. Exemplary biocompatible substrates of the implant composition include those that are natural or synthetic. Implant compositions may also comprise functional elements that may be constitutive or inducible through physical or chemical stimuli to up or down regulate the regulatory function during treatment. Preferably, the implant composition may be administered via gastrointestinal tract (as oral or suppository), parenteral (intramuscular, intravenous or subcutaneous) or topical, but is not limited to these examples.

The present invention also contemplates a composition comprising human amnion cells transformed with a vector and additional components. For example, additional components may include cells, genetically modified cells, proteins, peptides, nonprotein bioorganic substances, therapeutic agents (anti-inflammatory, antibiotic, antiviral, anti-neoplastic or antifungal) and combinations thereof. . In one embodiment the human amnion cells transformed with the vector may comprise at least one exogenous gene element encoding a glial or brain derived neurotrophic factor that can be used for the prevention or treatment of CNS disease.

The population of transformed human amnion cells or a composition thereof can be administered to a patient at an exemplary dosage, for example, in the range of about 10 ³ cells to about 10 ¹⁰ cells daily. However, the particular dosage employed may be varied or adjusted as appropriately considered by one of ordinary skill in the art. The dosage may depend on many factors including but not limited to the method of infusion, the needs of the patient and the severity of the disease to be treated. Determination of the appropriate dosage for a particular patient is also well known to those of ordinary skill in the art.

The invention may be more readily understood by reference to the drawings and examples as well as the following detailed description. The present invention provides a population of human amniotic cells, wherein each lentiviral vector comprises a lentiviral vector comprising at least one exogenous gene. Lentiviral vectors as used herein are agents that can deliver genes of interest into cells without degradation in all cells. Lentiviral vectors may also include promoters that express genes in cells, such as, for example, amniotic cells. Lentiviral vectors are based on the nucleic acid backbone of the virus of the Lentivirus family of viruses. Lentiviral vectors may also include first generation, second generation, and third generation lentiviral. In one embodiment, the lentiviral vector can provide gene delivery more efficiently than Ad and AAV. In addition, lentiviral vectors are capable of stably expressing external transplant genes without detectable pathogens or immunogenicity derived from viral proteins. Human amnion cells transformed with the lentiviral vectors according to the invention may be particularly efficient for gene therapy.

In one embodiment, the lentiviral vector is a third generation lentiviral. For example, third generation lentiviral has a lentiviral packaging gene isolated into at least three independent plasmids. In addition, lentiviral vectors, such as third generation lentiviruses, lack the HIV-1 tat gene (a strong transcription activator of the HIV-1 LTR promoter essential for viral replication) and accessory genes (vpr, vpu, vif and nef). There may be. Preferably, the EMV gene of HIV-1 can be substituted with the VSVG gene and the remaining HIV genome can be divided into two expression constructs. Exemplary lentiviruses may include PWPT, PLVTHM or PLV vectors.

Human amniotic cells arise from the initial internal cell mass of blastocysts about 8 days after fertilization. Human amnion cells can be obtained from human amnion. Exemplary human amnion cells include amnion epithelial and mesenchymal stem cells. Human amnion cells can express some markers normally present in stem cells, such as 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. In addition, evidence may suggest that HLA-A, HLA-B, HLA-C and HLA-DR antigens and β2 microglobulin on the surface of amnion cells are scarce or little expressed. Akle et al., Lancet, 1981, 2 (8254): 1003-5; Adinolfi et al., Nature, 1982, 295: 28. Human amnion cells according to the present invention can express HLA class lb (HLA-G) which can provide low immune rejection and long term survival in the host. have. Similarly, human amnion cells can be differentiated after xeno- or allo-transplantation. Ueta et al., Clin. Exp. Immunol., 2002, 129 (3): 464-70.

Promoters that regulate transcription from vectors in host cells in mammals can be obtained from a variety of sources such as, for example, RNAi-induced, Cre-rox P and doxycycline-induced systems. Vectors for transformation of human amnion cells can be designed to carry exogenous genetic elements that can be expressed by said cells. Exogenous genetic elements can include, for example, marker genes. Such marker genes can produce a product and can be used to determine whether a gene has been delivered to and expressed by a cell. Exemplary marker genes encode P-galactosidase and can include Escherichia coli's lacZ gene, green fluorescent protein (EGFP) or improved green fluorescent protein (GFP).

In one embodiment the exogenous gene element may also include a gene that replaces or supplements an original gene capable of treating central nervous system disease. For example, the exogenous gene element may be a neuronal growth factor, brain-derived neurotrophic factor, hypoxanthine guanine phosphoribosyltransferase, glial cell-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; Patella et al., Gene, 1989, 80: 137-44; Bjorklund et al, Brain Res., 2000, 886: 82-98; Kang et al., Hum. Cell, 2001, 14: 39-48; Yamada et al., Proc. Natl. Acad. Sci., 1999, 96: 4078; Tuszynski et al., Exp. Neurol., 1998, 154: 573-82; Saille et al., Neuroscience, 1999, 92: 1455-63; Haase et al., Ann. Neurol, 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 may comprise glial derived neurotrophic factor (GDNF) or brain derived neurotrophic factor (BDNF) genes.

Neurotrophic factor is responsible for the growth and survival of neurons during development as well as the maintenance of adult neurons. For example, models in zoology and in vitro show that certain neurotrophic factors allow damaged neurons to grow. In one embodiment the amniotic cell comprising a lentiviral vector comprising at least one exogenous gene element encoding 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. BDNF is widely and abundantly expressed in the central nervous system and is available in some peripheral nervous system neurons that consume neurotrophin produced by peripheral tissues. BDNF promotes survival and differentiation of certain neuronal populations during development.

During adulthood, BDNF can regulate the synaptic strength of neurons and is associated with hippocampal mechanisms of learning and memory as well as spinal cord mechanisms for pain. Many central nervous system disorders are also associated with reduced trophic support. Given that BDNF and its high affinity receptors are abundant throughout the central nervous system, BDNF can be a potent neuroprotective agent effective in the treatment of Parkinson's disease, Alzheimer's disease, depression, epilepsy and chronic pain, but is not limited to the examples above. no. Glioblastoma-derived neurotrophic factor, GDNF, also belongs to the family of neurotrophic factor proteins. GDNF promotes survival and morphological differentiation of dopaminergic neurons and increases dopamine uptake. GDNF can rescue motor neurons from programmed cell death and death by axotomy. GDNF is a particularly potent factor for the survival and axon growth of dopamine-activated neurons in the midbrain and has been shown to improve motor deficiency and reduce brain damage in many animal models. Bjorklund et al., Brain Res., 2000, 886: 82; Gash et al, Nature, 1996, 380: 252; Kordower et al., Science, 2000, 290: 767; Zu met al., Brain Res. Rev., 2001, 36: 222.

One example of an exogenous gene element includes said GDNF gene. 1 shows a lentiviral construct comprising a GDNF exogenous gene element. Exemplary exogenous gene elements as disclosed herein may include, but are not limited to, citation examples provided herein and sequences obtained from GenBank. In one embodiment the exogenous genetic element can be prepared or modified through traditional recombinant techniques.

Another aspect of the invention includes a composition comprising said human amnion cells and at least one pharmaceutically acceptable carrier. For example, human amnion cells and their compositions comprising lentiviral vectors with exogenous gene elements can be implanted into the body of an individual for gene therapy. Preferably, human amnion cells comprising a lentiviral vector with an exogenous gene element can be used for the treatment of brain neurological disease. Disorders of the central nervous system include damage to the brain, spinal cord, cranial nerves, nerve roots, and autonomic nervous system. Central nervous system diseases may also include, 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 of the brain do not receive enough blood to maintain normal nerve function. The condition may be the result of various diseases or arterial obstructions such as sympathetic strangulation. Cerebral ischemia can lead to the death of neurons and brain damage. In addition, brain bleeding leads to a lack of oxygen in the brain. A sharp loss of consciousness due to rupture or occlusion of blood vessels, and central nervous system trauma are damage to the brain and spinal cord resulting from indirect damage due to damage to the brain and spinal cord directly or surrounding bone, soft tissue or blood vessels.

Hereditary diseases of the nervous system include progressive damage to nerves such as, for example, Huntington's chorea (HD), cataract familial dementia (Tay-Sachs), plaque nucleus biliary atrophy (DRPLA), and wagon-Joseph disease (MJD). It is a hereditary, slowly progressing disease that can be caused by. Degenerative neurological disease is a disease caused by the deterioration of certain nerve cells (neurons). Changes in these cells cause the cells to function abnormally and eventually cause cell death. As well as multiple sclerosis, Alzheimer's disease, Parkinson's disease and Amyotrophic lateral sclerosis (ALS) are due to the degeneration of neurons in the central nervous system.

Neoplasms of the central nervous system include, but are not limited to, brain hemispheres, basal ganglia, hypothalamus, thalamus, brainstem and cerebellum. Cerebral neoplasms can be divided into primary forms (derived from brain tissue) and secondary forms (metastases). Primary neoplasms can also be divided into benign and malignant forms. In general, brain tumors can be classified by timing of initiation, tissue type or location of onset.

Spinal cord trauma is spinal cord injury resulting from a direct wound on the spinal cord itself or an indirect wound due to damage to bone, soft tissue, or blood vessels around the spinal cord. Parkinson's disease is a chronic progressive neurological disease linked to reduced dopamine production in melanoma and may be manifested by tremors and weakness in resting muscles as well as anxious shuffling gait. Symptoms of Parkinson's disease are caused by neuronal loss of a portion of the brain called medulla, which causes a decrease in dopamine (neurochemicals) throughout the brain. Such destruction occurs due to genetic or environmental factors and combinations thereof.

In one embodiment, intra-brain transplantation of GDNF transformed human amniotic cells (eg, epithelium) on ischemic rats made by intermediate cerebral artery occlusion (MCAo) can advantageously improve behavioral dysfunction and Infarction can be reduced. In addition, markers of neurons and neuronal stem cells can be detected in these transgenic human amnion cells. Similarly, many of these transgenic human amniotic cells remain alive and migrate to the infarct site.

For example, the BDNF gene, an important component of growth factors for the nervous system, plays a physiological role in the development of the central nervous system and regulation of mature neurons. The BDNF gene also provides strong neuroprotective effects of variously damaged neurons (regardless of the cause of the damage). In the brain, the BDNF protein of AD or PD patients is less than that of normal people. Intra-brain transplantation of BDNF transformed human amnion cells (eg epithelium) into PD individuals (rats) can significantly improve behavioral dysfunction and increase BDNF protein. Also according to the present invention, transplantation of BDNF transformed human amniotic cells into rhesus macaques with incomplete dorsal spinal cord injury can significantly improve behavioral dysfunction.

As used herein, "optional" or "optionally" may mean that an event or environment described later may or may not occur, and the description above may refer to the event. Or the environment includes examples that occur or do not occur.

As described, pharmaceutically acceptable carriers can include sterile aqueous solutions, suspensions, and emulsions. In addition, the aqueous solution carrier may include PBS or Hankas' solution, but is not limited to these examples. Exemplary emulsions or suspensions may include collagen, hydroxyproxy cellulose, microcrystalline cellulose, amylum, PVP, agar, pectin, magnesium aluminate silicate or magnesium aluminate.

The number of cells to be transfected can be appropriately determined according to the condition of the patient and the ability of the cells to produce the desired gene product according to the present invention. In one embodiment the number of cells to be transfected may be about 10 ⁵ to 10 ¹⁰ . The method of treatment also depends on the method of transplanting the cells and compositions thereof. In addition, treatment methods may include sterilization methods such as, for example, direct dosing or brain transplantation.

The following examples illustrate the benefits of the present invention, which have not been described previously, and are provided to further assist those skilled in the art to make and use such human amniotic cells. The above embodiments may include or include variants or embodiments of the present invention described above. Said embodiments as described above may further comprise or comprise variants of some or all of the other embodiments of the invention, respectively. In addition, the biological methods associated with traditional techniques are also described herein. Such traditional techniques are known in the art and are described, for example, in Molecular Cloning: A Laboratory Manual, 3rd Edition, vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2001 and Current Protocols in Molecular Biology, ed. Generally described in Ausubel et al., Greene Publishing and Wiley-Interscience, N. Y., 1992. Similarly, exemplary methods for chemical synthesis of nucleic acids are described in Beaucage et al., Terra. Letts., 1981, 22: 1859-12 and Matteucci et al., J. Am. Chem. Soc, 1981, 103: 3185, but is not limited to these examples. For example, chemical synthesis of nucleic acids can be performed using commercially automated oligonucleotide synthesizers. Immunological methods are also described in Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, N.Y., 1992, but are not limited to these examples. Traditional methods of gene delivery and gene therapy can also be adapted for use consistent 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 Human Gene Therapy, ed. Hodgson et al., Springer Verlag, 1996.

Other features and advantages of the invention will be apparent from the following detailed description, given in conjunction with the accompanying drawings.

1, a map of lentiviral vectors used herein. The vector is a central polypurine moiety that promotes transgene expression as well as regulatory and cis-acting elements after transcription of Wood-Neck hepatitis virus (WPRE or WHV). cPPT). All vectors include the extender 1-alpha promoter (EFl-α) or human cytomegalovirus (CMV) promoter, which is a strong transcription factor in most cell types.

2 Efficient delivery, integration and sustained long term expression of EGFP in human amnion cells. EGFP expression in human amnion cells 7 days after transformation with a lentiviral vector or DOTAP (FIG. 2A). EGFP was evaluated by photograph taken by fluorescence microscope (x10), and transformation efficiency was measured by FACS. EGFP expression was maintained for 5 weeks without apparent change (FIG. 2B). The EGFP was inserted into genomic DNA and transcribed into mRNA detected by PCR analysis (FIG. 2C). The same percentage of GO-Gl cells before and after infection with the vector was observed (FIG. 2D).

3, Lentiviral vector mediated siGFP inhibition in human amnion cells. The photographs were taken by fluorescence microscopy (x10) 7 days after transfection with EGFP expressing lentiviral vector (FIG. 3A) and the efficiency was measured by FACS (FIG. 3B). Seven days after transfection of human amniotic cells with an EGFP expressing lentiviral vector with a lentiviral vector expressing siGFP (FIG. 3C), the absence of EGFP expression was found only in the siGFP expression vector group. Inhibition of EGFP transcription was detected by RT-PCR analysis (FIG. 3D).

4, EGFP was efficiently regulated by the Cre-RoxP system based on lentiviral vectors in human amnion cells. EGFP was observed by fluorescence microscopy (× 10) after transformation with lenti-EGFP (FIG. 4A). Fluorescence was silenced by 7 days after cotransfection of Lenti-EGFP and Lenti-Cre (FIG. 4B), EGFP efficiency treated with Lenti-Cre was scored by FACS (FIG. 4C). PCR of genomic DNA demonstrated that EGFP was deleted in lenti-Cre (FIG. 4D), and the positive control was the result of RT-PCR of EGFP.

5, lentiviral vector mediated DOX induced gene expression. The EGFP was expressed in human amnion cells based on PLVTHM (FIG. 5A). The human amniotic cells were cotransfected with LVtTR-KRAB and PLVTHM (FIG. 5B) as well as with LVtTR-KRAB and PLVTHM in the presence of DOX (FIG. 5C). Human amnion cells were observed by fluorescence microscopy (× 10).

6, Expression of viral proteins GAG and EGFP in HeLa cells cultured in human amnion cells and human amnion cell condition medium. RT-PCR of EGFP showed that human amnion cells transformed with lenti-EGFP maintained EGFP expression, but the HeLa cells cultured in human amnion cell condition medium did not have EGFP expression (FIG. 6A). The structural gene GAG could not be detected in human amnion cells transformed with the lenti-EGFP or HeLa cells cultured in human amnion cell condition medium (FIG. 6B).

7, mean infarct volume in the experimental group. A decrease in the volume of ischemic injury was detected in the cell transplant group compared to the PBS group when measured 16 days after ischemia.

8, immunostaining of human amnion cells-GDNF in vivo. Immunohistochemical staining of GDNF from the brains of xenografted rats 14 days after transplantation revealed more GDNF positive human amniotic cells in the brains of the human amniotic cell-GDNF group (FIG. 8A). 8B shows a high performance micrograph showing the box portion of FIG. 8A. DAB and hematoxylin counter staining are shown in FIGS. 8C and 8D. As can be seen, there was no nestin expression in the opposite region.

FIG. 9, 3 weeks after transplantation, strong MAP2 expression was detected by immunohistochemistry (FIG. 9A) in the injection tract and by DAB and hematoxylin counter staining (FIG. 9C). 9B is the same site of opposite tissue. DAB and hematoxylin counter staining are shown in FIG. 9D.

10, nerve test.

11, beam-working test to detect a coordination function.

Example 1

Culture of Human Amniotic Cells.

Amnion membranes were mechanically stripped from the placenta chorion and extensively stripped off to remove underlying tissues (sponges and fibroblast layers) to obtain a pure epithelial layer with a basement membrane. The membrane was cut into segments with a razor. Sufficient enzyme solution was added to obtain signal cells. Human amnion cells were then cultured in cell culture medium.

vector.

Methods of making packaged retrovirus and lentiviral packaging systems are well known to those of ordinary skill in the art. Briefly, lentiviral vectors were prepared by transient transfection of 293T cells. 20 μg of lentiviral vectors (PWPT, LVTHM or PWPT-GDNF, FIG. 1), 10 μg of pMDlg / pRRE (or pCMVdR 8.2), 5 μg of pMD2 G and 5 μg of pRSV-REV were mixed and adjusted to 250 μl with water. It was mixed with 250 μl of CaCl ₂ 0.5M, and 500 μl of HeBS 2 × (0.28M NaCl, 0.05M HEPES, 1.5M Na ₂ HPO ₄ ) was added to the resulting mixture, and left for 30 minutes on the bench.

The dish was 5% CO ₂ It was left to stand in a 37 degreeC humid incubator which is atmosphere. The culture was aspirated. After 14 hours, 10 ml of fresh DMEM-10% FBS (PAA Austria) preheated to 37 ° C. was added gently and then incubated for 28 hours. The virus was collected and cleared by centrifugation at 1500 rpm for 15 minutes and filtered through a 0.45 μm filter. Ultracentrifugation was performed at 80,000 g and 4 ° C. for 90 minutes. Aliquots of the supernatants were resuspended as pellets in 1 ml PBS and stored at -80 ° C. The titration of the concentrated supernatant was performed via serial dilution of the vector stock on 1 × 10 ⁵ HeLa cells followed by fluorescence-activated flow measurements, ie Beckton Dickinson Immunocytometry Systems analysis. According to the formula 1 × 10 ⁵ HeLa cells × (%) EGFP positive cells × 1000 / μl virus, the titer of the lentiviral vector was calculated between 0.1-1 × 10 ⁹ TU / ml.

Human amniotic cells infected with lentiviral vectors.

The viral vector was added to amnion cells cultured for 2 days the next day in the presence of 1-10 μg / ml of polybrene (Sigma-Aldrich). After washing with PBS, culture was continued in cell culture medium so that exogenous gene elements could be introduced into human amnion cells.

Example 2

Lentivirus Vector Infection.

Research and use of the human amniotic membrane was approved by the Patient and Ethics Committee Reviews. For example, the amnion membrane is mechanically peeled from the placenta chorion obtained from a woman who has received a simple uncomplicated cesarean section, and the underlying tissue (cavernous or fibroblast layer) is obtained to obtain a pure epithelial layer with a basal membrane. Exfoliated extensively to remove. The membrane was placed in a 250 ml wide-mouthed flask containing RPMI 1640 medium and cut with a razor blade to make 0.5-1.0 pieces. 37 ° C. trypsin / EDTA solution was added to the culture to cover the membrane twice, first and second for about 30 and 15 minutes, respectively. The obtained cells were seeded in a 6 well plate of RPMI-1640 medium layer supplemented with 10% fetal calf serum (PAA Austria), streptomycin 100 μg / ml, penicillin 100 U / ml and glutamine 0.3 mg / ml, It was then incubated in a humid atmosphere of 5% CO ₂ in air at 37 ° C. Human amnion cells were plated in 6 well plates (2 × 10 ⁵ cells / well, Costar) and polybrene (Sigma-Aldrich) was brought to a final concentration of 8 μg / ml. After incubation for 48 hours, the lentiviral vector (PWPT) was added (MOI = 100). In addition, Boehringer Mannheim (DOTAP) was used to transfect human amnion cells.

Transformed human amnion cells were cultured for 1 week. EGFP expression was visualized by fluorescence microphotography and analyzed by fluorescence activated cell sorting (FACS). Genomic DNA was isolated, for example as described by Promega, and whole RNA isolation and reverse transcription was performed (Promega). To measure the relative expression of EGFP, semiquantitative PCR analysis of EGFP and β-actin (internal criteria) was performed by PCP amplification for 28 cycles. Primer sequences were EGFP (upstream 5'-cgagctggacggcgacgtaaac-3 'and downstream 5'-gcgcttctcgttggggtctttg-3') and β-actin (upstream 5'-aacgagcggttccgatgccctgag-3 'and downstream 5'-tgtcgccttcaccgttccagtt-3'). Amplification conditions at 94 ° C. for 5 minutes; 29 times for 1 minute at 94 ° C, 1 minute at 58 ° C, and 1 minute at 72 ° C; And at 72 ° C. for 10 minutes. The expected length of the EGFP RT-PCR product was 597 bp. The expression difference was normalized by each β-actin (590 bp). 10 ml from each RT-PCR product were loaded on a 1.5% agarose gel containing 0.5 μg / ml of ethidium bromide and separated by electrophoresis.

result.

EGFP can be observed by fluorescence microscopy 4 days after transformation. Human amnion cells as a control were transformed with DOTAP and these cells were scored by fluorescence activated cell sorting (FACS) on day 7. Transformation of the lentiviral vector detected that about 90% of the cells were successfully transformed with the lentiviral vector and more effective than DOTAP (FIG. 2A), and less than 5% (not shown) with DOTAP successfully. In turn, EGFP expression was measured continuously for 5 weeks and EGFP-positive human amniotic cells were maintained for the duration of the culture (FIG. 2B). To measure the insertion of EGFP into human amnion cells, genomic DNA and mRNA were extracted 7 days after transformation and PCR and RT-PCR amplification were performed. The result was that EGFP was inserted into the human amnion cell genome (FIG. 2C) and expressed stably at the mRNA level. The lentiviral vector did not affect the percentage of GO-Gl phase cells after transfection (FIG. 2D), demonstrating that human amnion cells modified with the lentiviral vector are stable gene messengers. As such, human amnion cells transformed with the lentiviral vectors of the present invention may provide stable gene delivery and may be useful for the prevention or treatment of CNS disease.

Example 3

Inhibition of siGFP mediated by lentiviral vectors in human amnion cells.

Human amnion cells were plated on 6 well plates (2 × 10 ⁵ cells / well, Costar) and polybrene (Sigma-Aldrich) was added to the wells. Lentivirus vectors (pLVTHM and pLVTHMsiGFP) were added at a final concentration of 8 μg / ml (MOI = 100). EGFP expression was visualized by fluorescence microphotography and analyzed by fluorescence-activated cell sorting (FACS). To measure the relative expression of EGFP, semi-quantitative PCR analysis of EGFP and β-actin (internal criteria) was performed by PCR amplification for 28 cycles. Primer sequences were EGFP (upstream 5′-cgagctggacggcgacgtaaac-3 ′ and downstream 5′-gcgcttctcgttggggtctttg-3 ′) and β-actin (upstream 5′-aacgagcggttccgatgccctgag-3 ′ and downstream 5′-tgtcgccttcaccgttccagtt-3 ′). Amplification conditions were 5 minutes at 94 ° C; 28 times for 1 minute at 94 ° C, 1 minute at 58 ° C, and 1 minute at 72 ° C; And at 72 ° C. for 10 minutes. The expected length of the product of the EGFP PCR was 597 bp. The expression difference was normalized by each β-actin (590 bp). 10 μl from each PCR product was loaded on a 1.5% agarose gel containing 0.5 μg / ml of ethidium bromide and separated by electrophoresis.

result.

RNA interference is the best gene-silencing process of RNA mediated gene regulation after transcription. Expression of siRNA delivered by lentiviral vectors can be used to functionally silence EGFP expression in human amnion cells. The sequence of siGFP in the lentiviral vector (PLVTHMsiGFP) was transcribed into dsRNA, which mediates sequence specific cleavage with the formation of ribonucleoprotein complexes. In the first experiment, the ability of the lentiviral vector PLVTHM to express EGFP in human amnion cells (Figure 3A) was identified. MOI = 100 assured a good rate of transformation. The human amnion cell-EGFP cells transformed with PLVTHM showed strong EGFP expression regardless of the culture conditions. In contrast, human amnion cell-siGFP cells showed strong downregulation of EGFP cotransformed with constitutively active PLVTHM and PLVTHMsiGFP (FIG. 3C). The results of FACS demonstrated that PLVTHMsiGFP silenced EGFP expression up to 15% in human amnion cell-siGFP cells (FIG. 3B). Based on the RT-PCR results, it was found that inhibition occurs at the mRNA level (FIG. 3D).

Example 4

Lentiviral vectors based on the Cre-RoxP system delete human amnion cells transformed with EGFP.

Human amnion cells were plated on 6 well plates (2 × 10 ⁵ cells / well, Costar) and polybrene (Sigma-Aldrich) was added to the wells. Lentivirus vectors (PWPT and PWPT-Cre) were added at a final concentration of 8 μg / ml (MOI = 100) and transformed human amniotic cells were incubated for 1 week. EGFP expression was shown by fluorescence micrographs and analyzed by fluorescence-activated cell sorting (FACS). Genomic DNA was isolated, for example, as described by Promega. To measure the relative expression of EGFP, semiquantitative PCR and analysis for EGFP and β-actin (internal criteria) were performed by PCP amplification for 28 cycles. Primer sequences were EGFP (upstream 5′-cgagctggacggcgacgtaaao-3 ′ and downstream 5′-gcgcttctcgttggggtctttg-3 ′) and β-actin (upstream 5′-aacgagcggttccgatgccctgag-3 ′ and downstream 5′-tgtcgccttcaccgttccagtt-3 ′). Amplification conditions were 5 minutes at 94 ° C; 28 times for 1 minute at 94 ° C, 1 minute at 58 ° C, and 1 minute at 72 ° C; And at 72 ° C. for 10 minutes. The expected length of the product of the EGFP PCR was 597 bp. The expression difference was normalized by each β-actin (590 bp). 10 μl from each PCR product was loaded on a 1.5% agarose gel containing 0.5 μg / ml of ethidium bromide and separated by electrophoresis.

Cre is a type of integrase family of site-specific recombinases that catalyzes recombination between loxP DNA elements. Sternberg et al., J. MoI. Biol., 1981, 150: 467-86. Attempts have been made for Cre to generate cell lines of human amniotic cells that catalyze the deletion of loxP-flanked specific genes based on lentiviral vectors. For example, there is loxP in the 3 'LTR of the lentiviral vector, and after reverse transcription, loxP was inserted at both ends of the LTR. Cre catalyzed the deletion of loxP- flanked lentiviral fragments in the genome of human amnion cells.

result.

FACS results demonstrated that EGFP was silenced by less than 10% (FIGS. 4A, 4B and 4C). In addition, it was also found that this deletion appeared in genomic DNA (FIG. 4D). As such, specific genes with PWPT-Cre in human amnion cells can be knocked out.

Example 5

Lentiviral vectors based on doxycycline-induced systems regulate EGFP expression.

Human amnion cells were plated on 6 well plates (2 × 10 ⁵ cells / well, Costar) and polybrene (Sigma-Aldrich) was added to the wells. At a final concentration of 8 μg / ml, lentiviral vectors (LVTHM and lenti-tTRKRAB) were added (MOI = 100). After 48 hours of incubation, doxycycline (final concentration 5 μg / ml, Sigma) was added to the co-transformed cells with PLVTHM and Lanti-tTRKRAB.

result.

During the absence of doxycycline, the tTR-KRAB protein binds specifically to tetO, providing a means for inhibiting the activity of the nearby promoter (up to 3 kb from the DNA binding site). In contrast, in the presence of doxycycline, tTR-KRAB is sequestered away from tetO to allow gene expression. Lentivirus vector mediated doxycycline inducible systems were tested for the doxycycline inducible regulation of EGFP in human amnion cells and then observed in human amnion cells transformed with PLVTHM (FIG. 5A). The tTRKRAB based on lentiviral vectors inhibited the expression of EGFP in the absence of doxycycline (FIG. 5B). In contrast, the addition of doxycycline to these dually transformed cells resulted in (re) expression of EGFP (FIG. 5C).

Example 6

Biosafety Detection

The cells were transformed with PWPT for 3 days and washed extensively to detect the inserted vector in human amnion cells. The medium was then collected and filtered through a 0.45 μm filter. HeLa cells with medium were incubated for 3 days and the genomic DNA of the HeLa cells and human amniotic cells were isolated as described, for example, by Promega. The genomic DNA (100 ng) then contains primers EGFP (upstream 5'-cgagctggacggcgacgtaaac-3 'and primers homologous to the HIV-I gag gene as well as primers (upstream 5'-gagtatctgatcatactgtcctac-3' and downstream 5'-ggaactactagtacccttcaggaa-3 ') PCR was performed using downstream 5′-gcgcttctcgttggggtctttg-3 ′ and β-actin (upstream 5′-aacgagcggttccgatgccctgag-3 ′ and downstream 5′-tgtcgccttcaccgttccagtt-3 ′). Amplification conditions were 5 minutes at 94 ° C; 1 time at 94 ° C., 1 minute at 58 ° C., 28 times at 1 minute at 72 ° C .; And at 72 ° C. for 10 minutes. The expected lengths of the EGFP and gag products were 597 bp and 912 bp, respectively. The expression difference was normalized by each β-actin (590 bp). 10 μl from each RT-PCR product was loaded onto a 1.5% agarose gel containing 0.5 μg / ml of ethidium bromide and separated by electrophoresis.

result.

It was found that EGFP is well inserted into human amnion cells and that EGFP is not present in the genome of HeLa cells (FIG. 6A). GAG, a structural protein of lentivirus, which may be essential for virus formation, was not detected in human amnion cells or HeLa cells expressing EGFP (FIG. 6B). These results demonstrate that human amniotic cells infected with lentiviral may have a check-up for biosafety prior to transplantation.

Example 7

Human amnion cell brain ischemic rats were infected and transplanted.

Lenti-GDNF (MOI = 50) was added to human amnion cells incubated with DMEM-F12 for 2 days the next day in the presence of 8 μg / ml polybrene (Sigma-Aldrich). After growing for 5 days in DMEM-F12 medium without fetal calf serum, human amniotic cells were washed with PBS and then incubated in 0.25% Trypsin / 0.05% DNaseI (Sigma) / PBS at 37 ° C. for 20 minutes. . The cells were washed 2-3 times with 0.05% DNaseI / PBS and mechanically disassembled to obtain a single cell suspension. Aliquots of cell suspensions were assessed with regard to cell viability (Trypan Blue) and concentration. The viability of the cell suspension before transplantation was at least 95%. Human amnion cells (human amniotic cells-GDNF) modified with 8 × 10 ⁵ GDNF in ⁵ μl of PBS using a stereotaxic frame (Narishige) and a 26-gauge Hamilton syringe (MCAo rats) Was injected into the right dorsolateral striatum. Markgraf et al., Brain Res., 1992, 575 (2): 238-46. Closely the injection was 4 mm below the skull surface, 1 mm posterior and 3 mm lateral to the parietal, close to the ischemic border zone for 10 minutes. From parietal to front-rear (AP) = -1mm, mid-side (ML) = 3mm and equi-fold (DV) = 4mm.

Three rats from each group were anesthetized and sacrificed with excess phenobarbital 16 days after surgery. The brain of the rat was carefully removed and cut into 2 mm pieces using a template. The pieces were stained with 2,3,4-triphenyltetrazolium chloride (TTC, 2% solution in PBS) for 30 minutes. We also photographed the pieces with the image acquisition system (Leica). Image analysis software AutoCAD (AutoDesk) was used to measure infarct volume. The result is expressed as a percentage of hemispheres.

result.

In all groups, infarct volume decreased from day 2 to day 16. There was no significant difference between the two groups after 2 days after MCAo. After 16 days after MCAo, there was a significant decrease in the percentage of infarct volume in the human amnion cell-GDNF group compared to rats in the control PBS group (FIG. 7).

Immunohistochemical Detection

Three days after transplantation, three rats of each group were killed for GDNF and MAP2 staining and examined by immunohistochemistry. The rat brain was fixed with 4% paraformaldehyde fixative for 2 days. After that, frozen sections of 30 μm (near the injection site) were cut in a cryostat at −20 ° C. and immunohistochemistry was performed. The sections were washed three times with PBS (pH 7.4). Endogenous peroxidase activity was inhibited with H ₂ O ₂ (0.3%) for 30 minutes. After blocking with 10% normal goat or horse serum at 4 ° C. overnight, the slide was a first antibody specific for MAP2 (1: 200, Sigma M4403) and GDNF (1: 100, Santa Cruz SC-9010) And incubated at 4 ° C. for 48 hours. The sections were then washed three times with PBS (pH 7.4), followed by biotin conjugated antibiotic mice or anti-rabbit IgG (Vector Laboratories). Sections were then washed with PBS and incubated with avidin-biotin-horseradish peroxidase complex. In addition, the preparation was stained using the vectortastein ABC kit (Vector Laboratories). Finally the slides were developed with diaminobenzidine (DAB). The immunohistochemical study was repeated at least three times.

result.

Human amnion cells engineered with GDNF (human amnion cells-GDNF) were injected into the lateral striatum and cortex of MCAo rats, and the implants were detected by immunohistochemistry in the brain of the subject. There was no positive signal for GDNF in the cortical region of normal non-transplanted rats (not shown). GDNF positive cells were detected at the injection tract of the brain of the ischemic rat, including the cortex and striatum. Many GDNF positive cells were also found in MCAo rats into which human amnion cells were transplanted (FIG. 8). After 16 days of transplantation, MAP2 positive cells were found at the injection site (FIG. 9), indicating that human amnion cells have the potential to differentiate into neurons.

Neurological investigation

Neurological findings were calculated on a modified scoring system developed by Longa et al. For example, a 0 indicates no neurological deficiency, a 1 indicates that the rat has difficulty stretching the opposite forelimb perfectly, and 2 indicates that the rat does not extend to the opposite forelimb. 3 means to draw a circle with a small angle to the other side, 4 means to draw a circle with a large angle to the other side, and 5 means to fall to the other side. The severity of nerve defects was observed in three seizure groups. Treatments were statistically investigated and analyzed by mean ± SE with a significance level of P <0.05.

Neurological defects were not found before MCAo. Rats of the seizure group (seizure with PBS and with human amniotic cells transformed with GDNF) were irradiated four times by day 16 after cell transplantation.

result.

The neurological findings scored on the 6 point scale were verified and the data showed significant differences between the two groups (FIG. 10). Follow-up comparison analysis revealed a significant difference (p <0.05) between human amnion cell-GDNF and PBS groups on day 4. These results show that human amnion cell-GDNF can significantly reduce the severity of neurological defects, particularly in early rats.

Mobility test (beam-walking test)

The beam-walking test was described by Ohlson et al. The beam is 1750 mm long and has an amplitude of 19 mm. The beam is placed 700 mm above the floor. The wall is alternately placed 2 cm around the beam (the rat will try to walk further when the wall is next to the beam). Scores range from 0 to 6. In particular, the 0 point is for the rat to fall over, the 1 point is for the rat to sit across the beam but sits still between the beams, the 2 point is for the rat to fall while walking, and the 3 point is for the rat to cross the beam. Although the affected hind limb cannot assist moving forward, 4 points are traversed by the rat moving more than 50% with the step of sliding the beam, and 5 points are caused by the rat's slight sliding. Steps traverse the beam, and 6 means the rat crosses the beam without a step of slipping. When the rat walks on the beam, such a score is scored. Treatments were statistically investigated and analyzed by mean ± SE at a significance level of P <0.05.

result.

The performance of the beam-walking test showed differences between the three animal groups at four time points from day 4 to day 16 post-transplant. Statistically significant improvement was detected in human amnion cell-GDNF transplantation over time (FIG. 11). In addition, the behavior of the human amnion cell-GDNF group was significantly better than the control at this early stage. Moreover, impaired coordination of untreated ischemic rats (control) did not recover.

These results showed that GDNF-producing human amnion cells can correctly repair the subject's defects after MCAo, and human amnion cells also play a significant role in subsequent recovery periods. These results may be caused by neurotrophic or anti-inflammatory factors secreted from human amniotic cells as well as differentiation into neurons.

Example 8

Preparation of Cell Injection

As described above, human amnion cell-GDNF was prepared and suspended in PBS.

Example 9

Transplantation of Human Amniotic Cells Infected with PLVTHM-BDNF in Rats with Parkinson's Disease

PLVTHM-BDNF (MOI = 100) was added to cells incubated with DMEM-F12 for 2 days the next day in the presence of 8 μg / ml polybrene (Sigma-Aldrich). After growing for 5 days in DMEM-F12 medium without fetal calf serum, human amniotic cells were washed with PBS and incubated in 0.25% trypsin (Sigma) /0.05% DNaseI (Sigma) / PBS at 37 ° C. for 20 minutes. . Cells were washed 2-3 times with 0.05% DNaseI / PBS and mechanically separated into single cell suspensions. The cell concentration was adjusted to 1-2 × 10 ⁸ . Experimental PD was produced in adult rats injected with the neurotoxin 6-hydroxydopamine in the brain. Toxins were injected into the medial forebrain bundles on one side of the rat brain according to the Orthotopic Guide. Three weeks later, the subjects were tested with apomorphine, which causes abnormal rotational behavior in implants with successful biochemical damage of the black-ancestor system. In the cell transplant group, human amnion cells modified with 8 × 10 ⁵ BDNF in ⁵ μl PBS were injected into the striatum of the 6-OHDA model of PD rats (AP = -5.0 mm, ML = ± 2.5 mm, DV = -6.5 mm). In the control group 5 μl PBS was injected into the striatum of the 6-OHDA model of PD rat (AP = -5.0 mm, ML = + 2.5 mm, DV = -6.5 mm). Thereafter, apomorphine induced rotations were observed after 2, 4 and 8 weeks.

result.

Human amnion cells infected with PLVTHM-BDNF in PD rats induced a significant decrease in apomorphine-induced rotation (Table 1).

Table 1 Comparison of Total Apomorphine-Turns in Two Groups

Time (week) Cell transplant group Control 0 260.2 ± 3.6 233.3 ± 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 10

Transplantation of PLVTHM-BDNF-infected Human Amniotic Cells into Rhesus Macaques with Incomplete Dorsal Spinal Cord Injury

PLVTHM-BDNF (MOI = 80) was added to cells incubated with DMEM-F12 for 2 days the next day in the presence of 8 μg / ml polybrene (Sigma-Aldrich). After 5 days of growth in DMEM-F12 medium without fetal calf serum, human amniotic cells are washed with PBS and incubated in 0.25% trypsin (Sigma) /0.05% DNaseI (Sigma) / PBS at 37 ° C. for 20 minutes. It was. The cells were washed 2-3 times with 0.05% DNaseI / PBS and mechanically separated into one cell suspension. The cell concentration was adjusted to 1-2 × 10 ⁸ . Rhesus monkeys with incomplete dorsal spinal cord injury were studied by the Tator method. Basso et al., J. Neurotrauma, 1995, 12 (1): 1-21. In the cell transplant group, human amnion cells transformed with 1xlO ⁷ BDNF were injected into the wound sites of two monkeys. In the control group, PBS was injected into the wound site of one monkey. Two months later, the BBB locomotor rating scale was evaluated. Basso et al., J. Neurotrauma, 1995, 12 (1): 1-21.

result.

The BBB score of the cell transplant group was 9.7. The BBB score of the control group was 5.2. As such, the human amniotic cells infected with PLVTHM-BDNF have been shown to improve hindlimb motor function in the subject.

 Although the present invention has been described herein in connection with preferred embodiments, those skilled in the art, after reading the foregoing, may make changes, substitutions of equivalents, and other forms of alteration. Each embodiment described above may include or be inserted into such a modification as disclosed in connection with some or all of the other embodiments. Accordingly, the protection afforded by the patents relating to the present invention is limited only by the scope and scope of the definitions contained in the appended claims and any equivalents thereof.

Claims (18)

A population of human amniotic cells comprising a lentiviral vector, wherein the lentiviral vector comprises an exogenous genetic element that can be expressed by the human amniotic cells. According to claim 1, wherein the exogenous gene elements are neuronal growth factor, brain-derived neurotrophic factor, hypoxanthine-guanine phosphoribosyltransferase, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase , A population of human amnion cells that encodes an aromatic L-amino acid decarboxylase, bcl-2 or tetrahydrobiopterin synthase. The population of human amniotic cells according to claim 1, wherein said exogenous gene element encodes a glial derived neurotrophic factor. The population of human amniotic cells according to claim 1, wherein said exogenous gene element encodes a brain derived neurotrophic factor. The method of claim 1, 2, 3 or 4, wherein the lentiviral vector is RNAi-inducible, Cre-loxP or doxycycline-induced system. A population of human amniotic cells comprising one or more regulatory transcriptional fragments of a doxycycline-inducible system. A composition comprising the population of human amnion cells of claim 1, 2, 3, 4 or 5 and a pharmaceutically acceptable carrier. A human amniotic cell comprising a lentiviral vector, wherein the lentiviral vector comprises an exogenous genetic element that can be expressed by said cell. 8. The method of claim 7, wherein the exogenous gene element is neuronal growth factor, brain-derived neurotrophic factor, hypoxanthine-guanine phosphoribosyltransferase, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase , Human amnion cells encoding an aromatic L-amino acid decarboxylase, bcl-2 or tetrahydrobiopterin synthase. A method of transducing a population of human amniotic cells with a lentiviral vector comprising culturing one or more lentiviral vectors with a population of human amniotic cells. A method of treating central nervous system disease in an individual using a population of human amniotic cells comprising a lentiviral vector with exogenous genetic elements. 11. The method of claim 10, wherein the central nervous system disease comprises cerebral ischemia, cerebral hemorrhage, central nervous system trauma, inherited diseases of the nervous system, neurodegenerative diseases or neoplasms of the central nervous system. The method of claim 10, wherein the central nervous system disease comprises cerebral ischemia, central nervous system trauma, or neurodegenerative disease. The method of claim 10, wherein the central nervous system disease is cerebral ischemia. The method of claim 10, wherein the central nervous system disease comprises spinal cord trauma. The method of claim 10, wherein the central nervous system disease is Parkinson's disease. A method for treating a central nervous system disease comprising requiring and administering to a subject an effective amount of a human amniotic cell comprising a lentiviral vector, wherein the lentiviral vector is an exogenous genetic element that can be expressed by said cell. Method comprising a. The method of claim 16, wherein the exogenous gene element is a neuronal growth factor, brain-derived neurotrophic factor, hypoxanthine-guanine phosphoribosyltransferase, glial cell-derived neurotrophic factor, ciliary neurotrophic factor, choline acetylase, tyrosine hydroxylase , Which encodes an aromatic L-amino acid decarboxylase, bcl-2 or tetrahydrobiopterin synthase The method of claim 16, wherein the central nervous system disease comprises cerebral ischemia, cerebral hemorrhage, central nervous system trauma, inherited diseases of the nervous system, neurodegenerative diseases or neoplasms of the central nervous system.

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