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993. Gene Delivery and Biodistribution in the Brain Following Adeno-Associated Virus Serotype 5 Vector-Mediated Gene Transfer
achieved by a bilateral intracerebral stereotaxic administration of an AAV-vector expressing antisense sequences linked to a modified U7 small nuclear RNA (snRNA). mRNA analyses and protein detection by immunohistochemistry and Western blotting were carried out on serial brain tissue sections. The DNA sequence confirmed that the 688 bp band resulting from RT-PCR corresponds to the exon 23-skipped mRNA. Moreover, this exon 23-skipped mRNA and the corresponding protein were selectively detected in a remote cerebral strucuture, the entorhinal cortex , directly connected to the hippocampus. These findings suggest a retrograde axonal transport of AAV particles to this afferent neuroanatomic area, such allowing the rescue of dystrophin expression in this neuronal netwotk. These results already suggest that mRNA manipulation may allow the reversion of genetic alterations in the mature brain. One main interest of this approach is the restoration of the lost protein not only in the target structure of the brain but also in the target cells , within the complex cell population of the brain tissue. Potentially, exon skipping may be used to selectively inactivate the expression of genes involved in cognitive processes and synaptic plast icity. It may open new opportunities for therapeutic strategies targeted to a functional neuroanatomical structure.
992. Ultrasound-Mediated Gene Transfer of HGF Improved Abeta-Induced Cognitive Impairment in Mice Associated with Upregulation of BDNF, Recovery of and Downregulation of Oxidative Stress Daisuke Takeuchi, I.2 Naoyuki Sato , I.2 Hitomi Kurinami, I.2 Munehisa Shimamura,' Shuko Takcda.P Mitsuru Shinohara, 1.2Toshio Ogihara.' Ryuichi Morishita.'
'Division ofClinical Gene Therapy, Osaka University; Suita, Osaka. Japan; 'Depanmem ofGeriatric Medicine. Osaka University. Suita, Osaka. Japan; 'Department ofAdvanced Clinical Science and Therapeutics. The University ofTokyo, TOkyo. Japan Background- Alzheimer disease (AD) is characterized by amyloid beta (AP) containing plaques in the neocortex and hippocampus and neurofibrially tangles, synaptic and neuronal loss along with progressive cognitive impairment. HOF (hepatocyte growth factor) is a unique multifunctional growth factor which can not only induce angiogenesis, but also act as a neurotrophic factor. Recently, we have reported that HGF improved memory and learning function after cerebral infarction in rats. In this study, we investigated whether HGF gene transfer could improve memory deficit in Ap-injection model. Methods ami Results- We used male ddY mice (6-8weeks old). AP( 1-40) was injected by intracerebroventricular (i.c.v.) administration into mice. 2 week after AP( 1-40) injection into cerebroventricle, mixture ofHGF gene/or BDNF gene and mierobubbles were injected into eerebroventricle. After the injection, ultrasonic irradiat ion was aplied to each mouse. I week after the administration ofHGF gene or BDNF gene , the water finding task and V-Maze test were carried out according to the established procedure. Mice treated with HGF gene or BDNF gene significantly had better performance in water finding task than mice treated with control vector. We confirmed upregulation ofBDNF after BDNF gene transfer, and unexpectedly found that HGF also upregulated BDNF in brain. We demonstrated FITC-albumin staining to evaluate the vessel density and confirmed that HGF and BDNF recovered the vessel density in dentate gyrus of hippocampus. We also demonstrated DHE staining to evaluate oxidative stress and confirmed that HGF reduced oxidative stress induced by Ap. Conclusions- Ultrasound-mediated gene transfer of HGF improved Ap-induced memory impairment in mice associated with upregulation ofBDNF, recovery of vessel density and downregulation of oxidative stress. HGF gene therapy might be a novel therapeutic strategy for Alzheimer's disease. Molecular Therapy Volume 15.Supplement 1. ~by Cop yright © The Americ...m Society o r Gene Therapy
2007
993. Gene Delivery and Biodistribution in the Brain Following Adeno-Associated Virus Serotype 5 Vector-Mediated Gene Transfer Lalitha Belur,' Zen hong Nan.? Zachary Dclvlorcst.! Chester B. Whitley,' Walter LOW,2 Carolyn Fairbanks; R. Scott Mcivor.'
'Dept. ofGenetics, Cell Biology and Development. University 0/ Minnesota. Minneapolis. AlN;'Dept. ofNeurosurgery, University ofMinnesota, Minneapolis. MN; 'Dept. ofPediatrics, University ofMinnesota, Minneapolis. MN; 'Dept. ofPharmacy, University ofMinnesota, Minneapolis. MN. Adeno-associated virus (AAV) is a replication deficient human parvovirus with a single stranded DNA genome and no known human pathogenicity. AAV vectors have been shown to be highly effective in mediating gene transfer and expression in different regions ofthe central nervous system in experimental animals. We recently reported successful use of an AAV2 vector transducing the human alpha-L-iduronidase gene for treatment ofa mouse model of mucopolysaceharidosis type I by intravenous injection ofthe vector at birth, resulting in prevention of metabolic, craniofacial and neurologic abnormalities exhibited in these mice (Molecular Therapy 9: 866, 2004). Our goal is to use AAV to deliver therapeutic genes to the mammalian brain and spinal cord ofadult MPS I mice as a model for gene therapy ofhuman MPS!. In order to increase vector distribution throughout the brain, we have examined the effect of mannitol infusion on patterns ofAAV serotype 5 transduction in the brain and spinal cord. Animals were injected intravenously with either mannitol or saline, followed by intracranial injection ofan AAV serotype 5 vector transducing GFP. Six weeks post-injection, animals injected intravenously with saline exhibited GFP expression that was limited to the choroid plexus proximal to the site of injection. However, in animals penneabilized with mannitol we observed widespread gene expression throughout the brain, including the posterior cortex, the hippocampus, the rostral migratory stream and the ventral anterior striatum. At 9 months post injection, we observed persistence of strong GFP expression in animals pre-treated with either mannitol or saline, with more widespread expression in animals infused with mannitol. Areas of expression in mannitol-permeabilized animals included the thalamus, striatum, entorhinal cortex, hippocampus and choroid plexus. These results provide evidence for the positive effects of mannitol infusion on long term GPP expression and distribution in the brain following a single intraventricular injection ofAAV5. We also studied the effect of mannitol infusion on gene delivery and biodistribution in the brain and spinal cord following intrathecal injection ofAAV5-GFP vector. Mice were infused with mannitol or saline and then they were injected with vector in the lumbar area. Animals sacrificed 6 weeks post-injection exhibited an increase in the level of Gf'P expression in the spinal cord when pre-infused with mannitol. A detailed analysis ofGFPexpression in the spinal cord and in the brain ofanimals after intrathecal injection of AAV vector is currently being undertaken. Results from these studies will provide the groundwork for future preclinical studies ofAAV vector mediated gene therapy ofMPSI by introduction of vector directly into the central nervous system.
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992. Ultrasound-Mediated Gene Transfer of HGF Improved Abeta-Induced Cognitive Impairment in Mice Associated with Upregulation of BDNF, Recovery of and Downregulation of Oxidative Stress Daisuke Takeuchi, I.2 Naoyuki Sato , I.2 Hitomi Kurinami, I.2 Munehisa Shimamura,' Shuko Takcda.P Mitsuru Shinohara, 1.2Toshio Ogihara.' Ryuichi Morishita.'
'Division ofClinical Gene Therapy, Osaka University; Suita, Osaka. Japan; 'Depanmem ofGeriatric Medicine. Osaka University. Suita, Osaka. Japan; 'Department ofAdvanced Clinical Science and Therapeutics. The University ofTokyo, TOkyo. Japan Background- Alzheimer disease (AD) is characterized by amyloid beta (AP) containing plaques in the neocortex and hippocampus and neurofibrially tangles, synaptic and neuronal loss along with progressive cognitive impairment. HOF (hepatocyte growth factor) is a unique multifunctional growth factor which can not only induce angiogenesis, but also act as a neurotrophic factor. Recently, we have reported that HGF improved memory and learning function after cerebral infarction in rats. In this study, we investigated whether HGF gene transfer could improve memory deficit in Ap-injection model. Methods ami Results- We used male ddY mice (6-8weeks old). AP( 1-40) was injected by intracerebroventricular (i.c.v.) administration into mice. 2 week after AP( 1-40) injection into cerebroventricle, mixture ofHGF gene/or BDNF gene and mierobubbles were injected into eerebroventricle. After the injection, ultrasonic irradiat ion was aplied to each mouse. I week after the administration ofHGF gene or BDNF gene , the water finding task and V-Maze test were carried out according to the established procedure. Mice treated with HGF gene or BDNF gene significantly had better performance in water finding task than mice treated with control vector. We confirmed upregulation ofBDNF after BDNF gene transfer, and unexpectedly found that HGF also upregulated BDNF in brain. We demonstrated FITC-albumin staining to evaluate the vessel density and confirmed that HGF and BDNF recovered the vessel density in dentate gyrus of hippocampus. We also demonstrated DHE staining to evaluate oxidative stress and confirmed that HGF reduced oxidative stress induced by Ap. Conclusions- Ultrasound-mediated gene transfer of HGF improved Ap-induced memory impairment in mice associated with upregulation ofBDNF, recovery of vessel density and downregulation of oxidative stress. HGF gene therapy might be a novel therapeutic strategy for Alzheimer's disease. Molecular Therapy Volume 15.Supplement 1. ~by Cop yright © The Americ...m Society o r Gene Therapy
2007
993. Gene Delivery and Biodistribution in the Brain Following Adeno-Associated Virus Serotype 5 Vector-Mediated Gene Transfer Lalitha Belur,' Zen hong Nan.? Zachary Dclvlorcst.! Chester B. Whitley,' Walter LOW,2 Carolyn Fairbanks; R. Scott Mcivor.'
'Dept. ofGenetics, Cell Biology and Development. University 0/ Minnesota. Minneapolis. AlN;'Dept. ofNeurosurgery, University ofMinnesota, Minneapolis. MN; 'Dept. ofPediatrics, University ofMinnesota, Minneapolis. MN; 'Dept. ofPharmacy, University ofMinnesota, Minneapolis. MN. Adeno-associated virus (AAV) is a replication deficient human parvovirus with a single stranded DNA genome and no known human pathogenicity. AAV vectors have been shown to be highly effective in mediating gene transfer and expression in different regions ofthe central nervous system in experimental animals. We recently reported successful use of an AAV2 vector transducing the human alpha-L-iduronidase gene for treatment ofa mouse model of mucopolysaceharidosis type I by intravenous injection ofthe vector at birth, resulting in prevention of metabolic, craniofacial and neurologic abnormalities exhibited in these mice (Molecular Therapy 9: 866, 2004). Our goal is to use AAV to deliver therapeutic genes to the mammalian brain and spinal cord ofadult MPS I mice as a model for gene therapy ofhuman MPS!. In order to increase vector distribution throughout the brain, we have examined the effect of mannitol infusion on patterns ofAAV serotype 5 transduction in the brain and spinal cord. Animals were injected intravenously with either mannitol or saline, followed by intracranial injection ofan AAV serotype 5 vector transducing GFP. Six weeks post-injection, animals injected intravenously with saline exhibited GFP expression that was limited to the choroid plexus proximal to the site of injection. However, in animals penneabilized with mannitol we observed widespread gene expression throughout the brain, including the posterior cortex, the hippocampus, the rostral migratory stream and the ventral anterior striatum. At 9 months post injection, we observed persistence of strong GFP expression in animals pre-treated with either mannitol or saline, with more widespread expression in animals infused with mannitol. Areas of expression in mannitol-permeabilized animals included the thalamus, striatum, entorhinal cortex, hippocampus and choroid plexus. These results provide evidence for the positive effects of mannitol infusion on long term GPP expression and distribution in the brain following a single intraventricular injection ofAAV5. We also studied the effect of mannitol infusion on gene delivery and biodistribution in the brain and spinal cord following intrathecal injection ofAAV5-GFP vector. Mice were infused with mannitol or saline and then they were injected with vector in the lumbar area. Animals sacrificed 6 weeks post-injection exhibited an increase in the level of Gf'P expression in the spinal cord when pre-infused with mannitol. A detailed analysis ofGFPexpression in the spinal cord and in the brain ofanimals after intrathecal injection of AAV vector is currently being undertaken. Results from these studies will provide the groundwork for future preclinical studies ofAAV vector mediated gene therapy ofMPSI by introduction of vector directly into the central nervous system.
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