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936. Intrathecal Delivery of the Sleeping Beauty Transposon System for Treatment of Chronic Pain
NEUROLOGIC & OPHTHALMIC II 936. Intrathecal Delivery of the Sleeping Beauty Transposon System for Treatment of Chronic Pain
Lalitha R. Belur,1 Carolyn Fairbanks,2 Lucy Vulchanova,3 Dan Schuster,4 Maureen Riedl,4 Kelly Podetz-Pedersen,1 Kelley F. Kitto,4 R. Scott McIvor.1 1 Dept. of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2Dept. of Pharmaceutics, University of Minnesota, Minneapolis, MN; 3Dept. of Veterinary Science, University of Minnesota, Minneapolis, MN; 4Dept. of Neuroscience, University of Minnesota, Minneapolis, MN.
Plasmid delivery systems have been successfully used to deliver genes into rodents intrathecally, although not as effectively as HSV or AAV-based viral vectors. The principal limitation with plasmid delivery is the transient duration of gene expression, which necessitates repeat administration of vector in order to achieve sustained expression of the therapeutic transgene. The Sleeping Beauty (SB) transposon system has been successfully used to mediate chromosomal transposition and thereby long-term expression in a variety of tissue types in vivo. Our goal is to use the SB vector system to deliver antinociceptive sequences to the dorsal root of the spinal cord and the dorsal root ganglia to modify nociceptive signaling. In order to optimize conditions for intrathecal delivery, we have constructed a transposon containing a bidirectional promoter consisting of a human elongation factor 1-alpha (EF1-alpha) promoter regulating expression of the green fluorescent protein gene and a phosphoglycerate kinase (PGK) promoter transcriptionally regulating expression of the firefly luciferase gene. These promoters are separated by a CpG-free synthetic enhancer. C57BL/6 mice were intravenously injected with mannitol in order to increase plasmid distribution throughout the spinal cord, followed by intrathecal administration of PEI-DNA complexes in the lumbar area. The PEIDNA complexes contained the transposon plasmid (10 and 50 µg) as well as varying amounts of SB transposase-encoding plasmid (0-50 µg) at an N/P ratio of 6. Animals are currently being evaluated for intensity and duration of luciferase gene expression using the Xenogen in vivo imaging system. In order to evaluate the cell types and subpopulations that are transfected, detailed analyses of GFP expression in the spinal cord and brain are also currently being undertaken. Results from these studies will provide a detailed characterization of SB-mediated gene transfer achieved after intrathecal delivery. This will enable intrathecal delivery of therapeutic sequences that are known to mediate spinal analgesia, with a goal toward attenuation of chronic pain, using a well-characterized non-viral integrating vector system µ.
937. Adeno-Associated Viral Vector (AAV)Mediated Gene Transfer in the Red Nucleus of the Adult Rat Brain: Comparative Analysis of the Transduction Properties of Seven AAV Serotypes and Lentiviral Vectors
Bas Blits,1 Sanne Derks,2 Jaap Twisk,2 Erich Ehlert,1 Jolanda Prins,1 Harald Petry,2 Joost Verhaagen.1 1 Neuroregeneration, Netherlands Institute for Neuroscience, Amsterdam, Netherlands; 2Research, Amsterdam Molecular Therapeutics (AMT), Amsterdam, Netherlands.
Recombinant adeno-associated viral vectors (AAV) are very promising gene transfer tools for the nervous system and gene therapy trials have begun in patients with enzyme deficiencies and neurodegenerative diseases. We have compared the efficiency of gene expression of seven AAV serotypes following a single injection in a major nucleus of the mid brain, the red nucleus, which is the origin of the rubrospinal tract. Injury to the rubrospinal tract is a commonly used model to study spinal cord regeneration. AAV serotypes 1-6 and 8 and a lentiviral vector were used, all encoding GFP under control S358
of the CMV promoter. AAV vectors were titermatched at 5 x 10E 11 GC/ml and 1 µl was injected into the red nucleus. The ratio of transduced neurons in the red nucleus at 1 and 4 weeks post-injection is presented in the figure below.
We found transduction efficiency differences per serotype and depending on the needs of transgene expression a decision of which serotype to use could be made. For transduction of the Red nucleus with the aim of overexpressing a transgene at high level for a longer period of time, serotype 1 is favored. Serotype 8 or 5 would be an option as well if onset of expression is preferred to be somewhat delayed. The use of lentiviral vectors should not be excluded either when also glial cells are required to be transduced. Serotypes 3 and 4 did not seem to transduce red nucleus neurons. Onset of expression was relatively fast using serotype 6 or lentiviral vectors. Transduction efficiencies were confirmed by quantification of the number of transduced RST fibers in the spinal cord. These data should be critical in designing an experiment that aims at studying the regenerative capacities of injured red nucleus neurons by transducing these neurons with genes that are associated with regeneration.
938. Preparation of Neurotrophic Factor Gene Expressing Collagen Devise for Retinal Diseases
Toshiaki Abe,1 Yumi Ishikawa,1 Ryosuke Wakusawa,1 Hideyuki Onami,1 Nobuhiro Nagai.1 1 Division of Clinical Cell Therapy, Tohoku University, Graduate School of Medicine, Sendai, Miyagi, Japan.
Purpose: To make new devise that can implant into or on the sclera for retinal protection. The devise will be made from collagen and include tetracycline derivatives (DOX)-inducible neurotrophic factor gene expressing cells. Methods: We constructed green fluorescein protein (GFP) or brain-derived neurotrophic factor (BDNF) gene into Tet-on vector and transduced into retinal pigment epithelial cells (RPE). We transplanted these cells into rat subretinal space or into sclera and confirmed weather these cells expressed the target genes by DOX eye drops. Then we tried to make collagen-based device with variable temperature-dependent bridging. The DOX inducible cells were cultured in the devises by variable methods such as direct cell drop, injection by Hamilton syringe, with temperature-dependent collagen or fibrin gel. The target gene expression was examined by western blotting, ELISA, or GFP expression. Cell viability was monitored by MTS and examined histologically. Some of the devices were implanted into the rabbit sclera and examined histologically. Results: GFP expression was monitored by DOX eye drops after subretinal or intra scleral injectionof the cells. The devises were maintained in the culture medium for more than one year. Direct cell culture on the devise showed different cell population in the devise. The cells with fibrin gel showed better cell proportion in the devises Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
Lalitha R. Belur,1 Carolyn Fairbanks,2 Lucy Vulchanova,3 Dan Schuster,4 Maureen Riedl,4 Kelly Podetz-Pedersen,1 Kelley F. Kitto,4 R. Scott McIvor.1 1 Dept. of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2Dept. of Pharmaceutics, University of Minnesota, Minneapolis, MN; 3Dept. of Veterinary Science, University of Minnesota, Minneapolis, MN; 4Dept. of Neuroscience, University of Minnesota, Minneapolis, MN.
Plasmid delivery systems have been successfully used to deliver genes into rodents intrathecally, although not as effectively as HSV or AAV-based viral vectors. The principal limitation with plasmid delivery is the transient duration of gene expression, which necessitates repeat administration of vector in order to achieve sustained expression of the therapeutic transgene. The Sleeping Beauty (SB) transposon system has been successfully used to mediate chromosomal transposition and thereby long-term expression in a variety of tissue types in vivo. Our goal is to use the SB vector system to deliver antinociceptive sequences to the dorsal root of the spinal cord and the dorsal root ganglia to modify nociceptive signaling. In order to optimize conditions for intrathecal delivery, we have constructed a transposon containing a bidirectional promoter consisting of a human elongation factor 1-alpha (EF1-alpha) promoter regulating expression of the green fluorescent protein gene and a phosphoglycerate kinase (PGK) promoter transcriptionally regulating expression of the firefly luciferase gene. These promoters are separated by a CpG-free synthetic enhancer. C57BL/6 mice were intravenously injected with mannitol in order to increase plasmid distribution throughout the spinal cord, followed by intrathecal administration of PEI-DNA complexes in the lumbar area. The PEIDNA complexes contained the transposon plasmid (10 and 50 µg) as well as varying amounts of SB transposase-encoding plasmid (0-50 µg) at an N/P ratio of 6. Animals are currently being evaluated for intensity and duration of luciferase gene expression using the Xenogen in vivo imaging system. In order to evaluate the cell types and subpopulations that are transfected, detailed analyses of GFP expression in the spinal cord and brain are also currently being undertaken. Results from these studies will provide a detailed characterization of SB-mediated gene transfer achieved after intrathecal delivery. This will enable intrathecal delivery of therapeutic sequences that are known to mediate spinal analgesia, with a goal toward attenuation of chronic pain, using a well-characterized non-viral integrating vector system µ.
937. Adeno-Associated Viral Vector (AAV)Mediated Gene Transfer in the Red Nucleus of the Adult Rat Brain: Comparative Analysis of the Transduction Properties of Seven AAV Serotypes and Lentiviral Vectors
Bas Blits,1 Sanne Derks,2 Jaap Twisk,2 Erich Ehlert,1 Jolanda Prins,1 Harald Petry,2 Joost Verhaagen.1 1 Neuroregeneration, Netherlands Institute for Neuroscience, Amsterdam, Netherlands; 2Research, Amsterdam Molecular Therapeutics (AMT), Amsterdam, Netherlands.
Recombinant adeno-associated viral vectors (AAV) are very promising gene transfer tools for the nervous system and gene therapy trials have begun in patients with enzyme deficiencies and neurodegenerative diseases. We have compared the efficiency of gene expression of seven AAV serotypes following a single injection in a major nucleus of the mid brain, the red nucleus, which is the origin of the rubrospinal tract. Injury to the rubrospinal tract is a commonly used model to study spinal cord regeneration. AAV serotypes 1-6 and 8 and a lentiviral vector were used, all encoding GFP under control S358
of the CMV promoter. AAV vectors were titermatched at 5 x 10E 11 GC/ml and 1 µl was injected into the red nucleus. The ratio of transduced neurons in the red nucleus at 1 and 4 weeks post-injection is presented in the figure below.
We found transduction efficiency differences per serotype and depending on the needs of transgene expression a decision of which serotype to use could be made. For transduction of the Red nucleus with the aim of overexpressing a transgene at high level for a longer period of time, serotype 1 is favored. Serotype 8 or 5 would be an option as well if onset of expression is preferred to be somewhat delayed. The use of lentiviral vectors should not be excluded either when also glial cells are required to be transduced. Serotypes 3 and 4 did not seem to transduce red nucleus neurons. Onset of expression was relatively fast using serotype 6 or lentiviral vectors. Transduction efficiencies were confirmed by quantification of the number of transduced RST fibers in the spinal cord. These data should be critical in designing an experiment that aims at studying the regenerative capacities of injured red nucleus neurons by transducing these neurons with genes that are associated with regeneration.
938. Preparation of Neurotrophic Factor Gene Expressing Collagen Devise for Retinal Diseases
Toshiaki Abe,1 Yumi Ishikawa,1 Ryosuke Wakusawa,1 Hideyuki Onami,1 Nobuhiro Nagai.1 1 Division of Clinical Cell Therapy, Tohoku University, Graduate School of Medicine, Sendai, Miyagi, Japan.
Purpose: To make new devise that can implant into or on the sclera for retinal protection. The devise will be made from collagen and include tetracycline derivatives (DOX)-inducible neurotrophic factor gene expressing cells. Methods: We constructed green fluorescein protein (GFP) or brain-derived neurotrophic factor (BDNF) gene into Tet-on vector and transduced into retinal pigment epithelial cells (RPE). We transplanted these cells into rat subretinal space or into sclera and confirmed weather these cells expressed the target genes by DOX eye drops. Then we tried to make collagen-based device with variable temperature-dependent bridging. The DOX inducible cells were cultured in the devises by variable methods such as direct cell drop, injection by Hamilton syringe, with temperature-dependent collagen or fibrin gel. The target gene expression was examined by western blotting, ELISA, or GFP expression. Cell viability was monitored by MTS and examined histologically. Some of the devices were implanted into the rabbit sclera and examined histologically. Results: GFP expression was monitored by DOX eye drops after subretinal or intra scleral injectionof the cells. The devises were maintained in the culture medium for more than one year. Direct cell culture on the devise showed different cell population in the devise. The cells with fibrin gel showed better cell proportion in the devises Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy