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  1. Article ; Online: Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

    Tadokoro, Takahiro / Miyanohara, Atsushi / Navarro, Michael / Kamizato, Kota / Juhas, Stefan / Juhasova, Jana / Marsala, Silvia / Platoshyn, Oleksandr / Curtis, Erik / Gabel, Brandon / Ciacci, Joseph / Lukacova, Nada / Bimbova, Katarina / Marsala, Martin

    Journal of visualized experiments : JoVE

    2017  , Issue 125

    Abstract: The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene ... ...

    Abstract The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 10
    MeSH term(s) Animals ; Brain/metabolism ; Dependovirus/genetics ; Female ; Genetic Vectors/genetics ; Genetic Vectors/metabolism ; Green Fluorescent Proteins/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; Spinal Cord/metabolism ; Video Recording
    Chemical Substances Green Fluorescent Proteins (147336-22-9)
    Language English
    Publishing date 2017-07-13
    Publishing country United States
    Document type Journal Article ; Video-Audio Media
    ISSN 1940-087X
    ISSN (online) 1940-087X
    DOI 10.3791/55770
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Subpial adeno-associated virus 9 (aav9) vector delivery in adult mice

    Tadokoro, Takahiro / Miyanohara, Atsushi / Navarro, Michael / Kamizato, Kota / Juhas, Stefan / Juhasova, Jana / Marsala, Silvia / Platoshyn, Oleksandr / Curtis, Erik / Gabel, Brandon / Ciacci, Joseph / Lukacova, Nada / Bimbova, Katarina / Marsala, Martin

    Journal of visualized experiments. 2017 July 13, , no. 125

    2017  

    Abstract: The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene ... ...

    Abstract The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 μL; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.
    Keywords Dependoparvovirus ; adults ; animal injuries ; animal models ; axons ; catheters ; gene expression ; genome ; green fluorescent protein ; mice ; motor cortex ; neurodegenerative diseases ; polyethylene ; rats ; spinal cord ; swine ; transgenic animals
    Language English
    Dates of publication 2017-0713
    Size p. e55770.
    Publishing place Journal of Visualized Experiments
    Document type Article
    ZDB-ID 2259946-0
    ISSN 1940-087X
    ISSN 1940-087X
    DOI 10.3791/55770
    Database NAL-Catalogue (AGRICOLA)

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  3. Article: Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.

    Miyanohara, Atsushi / Kamizato, Kota / Juhas, Stefan / Juhasova, Jana / Navarro, Michael / Marsala, Silvia / Lukacova, Nada / Hruska-Plochan, Marian / Curtis, Erik / Gabel, Brandon / Ciacci, Joseph / Ahrens, Eric T / Kaspar, Brian K / Cleveland, Don / Marsala, Martin

    Molecular therapy. Methods & clinical development

    2016  Volume 3, Page(s) 16046

    Abstract: Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult ... ...

    Abstract Effective in vivo use of adeno-associated virus (AAV)-based vectors to achieve gene-specific silencing or upregulation in the central nervous system has been limited by the inability to provide more than limited deep parenchymal expression in adult animals using delivery routes with the most clinical relevance (intravenous or intrathecal). Here, we demonstrate that the spinal pia membrane represents the primary barrier limiting effective AAV9 penetration into the spinal parenchyma after intrathecal AAV9 delivery. We develop a novel subpial AAV9 delivery technique and AAV9-dextran formulation. We use these in adult rats and pigs to show (i) potent spinal parenchymal transgene expression in white and gray matter including neurons, glial and endothelial cells after single bolus subpial AAV9 delivery; (ii) delivery to almost all apparent descending motor axons throughout the length of the spinal cord after cervical or thoracic subpial AAV9 injection; (iii) potent retrograde transgene expression in brain motor centers (motor cortex and brain stem); and (iv) the relative safety of this approach by defining normal neurological function for up to 6 months after AAV9 delivery. Thus, subpial delivery of AAV9 enables gene-based therapies with a wide range of potential experimental and clinical utilizations in adult animals and human patients.
    Language English
    Publishing date 2016-07-13
    Publishing country United States
    Document type Journal Article
    ZDB-ID 2872938-9
    ISSN 2329-0501 ; 2329-0501
    ISSN (online) 2329-0501
    ISSN 2329-0501
    DOI 10.1038/mtm.2016.46
    Database MEDical Literature Analysis and Retrieval System OnLINE

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