WO2002088337A1 - Nevroglie olfactive enveloppante immortalisee par l'introduction d'une telomerase - Google Patents

Nevroglie olfactive enveloppante immortalisee par l'introduction d'une telomerase Download PDF

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Publication number
WO2002088337A1
WO2002088337A1 PCT/ES2002/000191 ES0200191W WO02088337A1 WO 2002088337 A1 WO2002088337 A1 WO 2002088337A1 ES 0200191 W ES0200191 W ES 0200191W WO 02088337 A1 WO02088337 A1 WO 02088337A1
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WIPO (PCT)
Prior art keywords
oeg
cells
telomerase
nervous system
express telomerase
Prior art date
Application number
PCT/ES2002/000191
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English (en)
Spanish (es)
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WO2002088337A9 (fr
WO2002088337B1 (fr
Inventor
María Paz RUBIO RODRÍGUEZ
María Almudena RAMÓN CUETO
María Antonia Blasco Marhuenda
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Consejo Superior De Investigaciones Científicas
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Publication of WO2002088337A1 publication Critical patent/WO2002088337A1/fr
Publication of WO2002088337B1 publication Critical patent/WO2002088337B1/fr
Publication of WO2002088337A9 publication Critical patent/WO2002088337A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/04—Immortalised cells

Definitions

  • SNP peripheral nervous
  • CNS central nervous system
  • glial cells oligodendroglia, astroglia and microglia
  • axons of the sectioned olfactory neurons regenerate and restore the histological and functional integrity of the system.
  • OEG olfactory envelope glia
  • SUBSTITUTE SHEET (RULE 26) (ascending), nor can the motor orders generated by the brain be transmitted to the effector structures (descending information), with a loss of sensation and a paralysis below the level of the lesion.
  • peripheral nerve grafts (1, 20, 21), embryonic nerve tissue (22), infusions of neurotrophic factors and / or adhesion molecules (23, 24, 25, have been placed at the site of the lesion) 26), Schwann cell transplants (24, 26, 27), genetically modified cell transplants to produce axonal regeneration-promoting molecules (28), etc.
  • injured spinal axons grew within the multiple implanted permissive environments, but were unable to leave them and could not regenerate in the CNS inhibitory environment. This fact prevented that the lost histological integrity could be restored and therefore, the function of the damaged system.
  • Fibroblast genetically modified to produce nerve growth factor induce robust neuritic ingrowth after grafting to the spinal cord.
  • OEG olfactory envelope glia
  • telomerase By modifying the OEG with telomerase, it is intended to increase its division capacity in order to have, in the future, an OEG bank stored in hospitals, which can be used as therapy in nervous system injuries, simplifying, in addition, The procedure of obtaining OEG and guaranteeing its availability at the time it is necessary.
  • these cells could be used in the biochemical and molecular characterization of the envelope glia and in the purification of relevant molecules produced by the OEG.
  • These molecules could constitute pharmacological targets in the future in the treatment of nervous system lesions. They are subject to the patent we request:
  • telomere any molecule or molecules produced by the genetically modified OEG to express telomerase, or resulting from telomerase activity, either alone or in combination with other repair strategies, to treat diseases and lesions of the mammalian nervous system, including Primates, as well as any other pathology.
  • Phoenix packaging cells are used, which, by the conventional calcium phosphate method, are transfected to introduce the gene encoding the protein component of ribonucleoprotein telomerase.
  • the plasmid pBabe-mTert is used, which contains said gene instead of the "env, pol and gag" sequences of the retrovirus.
  • This plasmid also contains the puromycin resistance gene.
  • the next day medium is added with dexamethasone and sodium butyrate.
  • the supernatants are collected and titrated to calculate the retroviral vector production of these cells, by puromycin-resistant clone formation assay.
  • OEG cultures are obtained from olfactory bulbs of adult rats, as described in previous studies (36-39). To infect the OEG, and thus transfer the gene of interest, these cells are incubated with the supernatants obtained from the transfected Phoenix cells, and which contain the defective retrovirus, in the presence of polybrene and mitogens that stimulate the division of the OEG. The multiplicity of infection (mdi) remains close to or above 5. These infections are repeated in several successive rounds.
  • the cells are incubated with puromycin, at the dose that ensures that all non-transfected cells die within 4-6 days. After this time, the cells that survive are those that have been infected and, therefore, also express the m-Tert gene.
  • FIG. 1A An OEG clone containing m-Tert is shown in Figure 1-A, after selecting the cells by puromycin incubation.
  • the genetically modified OEG with m-Tert retains the typical morphology of the unmodified OEG.
  • Figure 1-B shows blue-stained OEG cells, which have been infected with supernatants obtained from Phoenix cells after transfection with pBabe-lacZ. Calibration bar in Figure 1-A and B: 35 Dm.
  • the genetically modified olfactory envelope (OEG) to express telomerase can be used to repair lesions of the mammalian nervous system including primates. These cells and / or their derived molecules may be used alone or in combination with other repair strategies. In addition to being used in spinal cord injuries, its use can be extended to all those nervous system pathologies that require axonal growth for healing. For example, they could be used in traumatic or ischemic lesions of other regions of the nervous system (optic nerve, etc.), in the treatment of neurodegenerative diseases such as Parkinson's disease, Huntington, some retinal degenerations, some ataxia, etc.
  • neurodegenerative diseases such as Parkinson's disease, Huntington, some retinal degenerations, some ataxia, etc.
  • telomeres can be combined with neural transplants (neuroblasts, stem cells, genetically modified neurons, etc.), to stimulate axonal growth in transplanted or host neurons. Since the introduction of telomerase in cells does not alter its phenotype or function, the OEG that expresses telomerase will also be used for cell, molecular and biochemical studies both in vitro and in vivo.

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention porte sur l'utilisation d'une névroglie olfactive enveloppante immortalisée par une télomérase ou sur l'utilisation d'une ou deux molécule(s) quelconque(s) produite(s) par celle-ci, soit seule ou en combinaison avec d'autres stratégies de réparation, afin de traiter des lésions du système nerveux chez les mammifères, y compris les primates.
PCT/ES2002/000191 2001-04-19 2002-04-18 Nevroglie olfactive enveloppante immortalisee par l'introduction d'une telomerase WO2002088337A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES200100909A ES2190336B1 (es) 2001-04-19 2001-04-19 Glia envolvente olfatoria modificada geneticamente mediante introduccion de telomerasa.
ESP200100909 2001-04-19

Publications (3)

Publication Number Publication Date
WO2002088337A1 true WO2002088337A1 (fr) 2002-11-07
WO2002088337B1 WO2002088337B1 (fr) 2003-03-06
WO2002088337A9 WO2002088337A9 (fr) 2004-05-21

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ES (1) ES2190336B1 (fr)
WO (1) WO2002088337A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005012513A1 (fr) * 2003-07-18 2005-02-10 Consejo Superior De Investigaciones Cientificas Nevroglies enveloppantes olfactives immortalisees de maniere reversible et leur utilisation dans la promotion de la regeneration neuronale
WO2012164137A1 (fr) 2011-05-30 2012-12-06 Fundación Investigación En Regeneración Del Sistema Nervioso Cellules mères et cellules de la souche neurale dérivées de la glie olfactive enveloppante, et leurs applications

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006631A1 (fr) * 1989-10-27 1991-05-16 Case Western Reserve University Activation de la regeneration de cns par la nevroglie du bulbe olfactif

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991006631A1 (fr) * 1989-10-27 1991-05-16 Case Western Reserve University Activation de la regeneration de cns par la nevroglie du bulbe olfactif

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DELUCIA T.M.: "Neurotrophic properties of an olfactory ensheating cell line", FASEB JOURNAL, vol. 16, no. 5, 22 March 2002 (2002-03-22), ANNUAL MEETING OF PROFESSIONAL RESEARCH SCIENTISTS ON EXPERIMENTAL BIOLOGY NEW ORLEANS, LOUISIANA, USA 20-24 APRIL 2002, pages A751 *
DOUCETTE R.: "Olfactory ensheating cells: past, present and future", FASEB JOURNAL, vol. 16, no. 5, 20 March 2002 (2002-03-20), ANNUAL MEETING OF PROFESSIONAL RESEARCH SCIENTISTS ON EXPERIMENTAL BIOLOGY NEW ORLEANS, LOUISIANA, USA 20-24 APRIL 2002, pages A751 *
FRANKLIN R.J.M.: "The remyelinating properties of transplanted olfactory ensheating cells", FASEB JOURNAL, vol. 16, no. 5, 22 March 2002 (2002-03-22), ANNUAL MEETING OF PROFESSIONAL RESEARCH SCIENTISTS ON EXPERIMENTAL BIOLOGY NEW ORLEANS, LOUISIANA, USA 20-24 APRIL 2002, pages A751 *
RAMON-CUETO A. ET AL.: "Functional recovery of paraplegic rats and motor axon regeneration in their spinal cords by olfactory ensheating glia", NEURON, vol. 25, no. 2, February 2000 (2000-02-01), pages 425 - 435 *
RAMON-CUETO A: "Olfactory ensheating glia transplants to repair spinal cord traumatic injuries in adult mammals", FASEB JOURNAL, vol. 16, no. 5, 22 March 2002 (2002-03-22), ANNUAL MEETING OF PROFESSIONAL RESEARCH SCIENTISTS ON EXPERIMENTAL BIOLOGY NEW ORLEANS, LOUISIANA, USA 20-24 APRIL 2002, pages A751 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005012513A1 (fr) * 2003-07-18 2005-02-10 Consejo Superior De Investigaciones Cientificas Nevroglies enveloppantes olfactives immortalisees de maniere reversible et leur utilisation dans la promotion de la regeneration neuronale
WO2012164137A1 (fr) 2011-05-30 2012-12-06 Fundación Investigación En Regeneración Del Sistema Nervioso Cellules mères et cellules de la souche neurale dérivées de la glie olfactive enveloppante, et leurs applications

Also Published As

Publication number Publication date
ES2190336A1 (es) 2003-07-16
WO2002088337A9 (fr) 2004-05-21
ES2190336B1 (es) 2005-02-01
WO2002088337B1 (fr) 2003-03-06

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