- Email: [email protected]
600. Herpes Simplex Virus 2 Gene, ICP10PK, Protects Against Neuronal Cell Death Associated with Valproic Acid-Induced Autism Spectrum Disorder
NEUROLOGIC GENE & CELL THERAPY II (PBWC) that relate to the disease pathogenesis. Genome-wide gene expression microarrays on pooled RNA samples showed significant dysregulation of >700 genes in PBWC in MPS IIIB mice. These results reflect the complexity of MPS IIIB pathology, including previously reported neurodegeneration, inflammation, oxidative stress, autophagy, and metabolic abnormalities, as well as currently unexplored disease aspects. Many of the dysregulated genes are considered to be tissue-specific, and were not anticipated to be dysregulated in PBWC. Further analyses of multiple genes linked to major pathways of neurodegeneration demonstrated a strong temporal brain-PBWC association in amyloidosis, synucleinopathy and prion protein (Prnp) deposition in MPS IIIB mice. A single systemic delivery of rAAV9-hNAGLU vector mediated efficient CNS and somatic restoration of NAGLU activity, and clearance of lysosomal storage pathology. Importantly, the treatment also led to correction of impaired neurodegenerative molecular pathways in both the brain and PBWC. Our data demonstrate that molecular changes in PBWC may reflect pathological status in the CNS and respond to rAAV9-hNAGLU gene delivery. Our approach may provide useful and accessible CNS-specific biomarkers for the evaluation of MPS IIIB disease progression and therapeutic treatment.
598. The Therapeutic Potential of MicroRNA Regulation in a Rat Intracerebral Hemorrhage Model
Jangsup Moon,1 Jeong-Min Kim,1 Jung Ick Byun,1 Kon Chu,1 Keun-Hwa Jung,1 Soon-Tae Lee,1 Jin-Hee Kim,2 Dong-Kyu Park,2 So-Yoon Kim,2 Jae-Kyu Roh.1 1 Department of Neurology, Seoul National University Hospital, Seoul, Republic of Korea; 2Laboratory for Neurotherapeutics, Seoul National University College of Medicine, Seoul, Republic of Korea. Background: MicroRNAs (miRNA) are short RNA sequences which regulate gene expression by modulating post-transcriptional process, which are involved in multiple signal pathways including neuronal development and injury. Intracerebral hemorrhage is one of the most devastating diseases with significant morbidity, but treatment option is still limited. We tried to evaluate potential therapeutic target of hemorrhagic stroke by miRNA analysis. Methods: We harvested hemorrhagic brain after 24 hours from a collagenase-induced rat ICH model and extracted RNA samples from each brain hemisphere. We performed miRNAs microarray analysis to compare their expression pattern difference between left hemorrhagic hemisphere and contralateral right hemisphere, and their expression levels were confirmed by quantitative real time polymerase chain reaction. We selected miRNAs for the therapeutic target study after reviewing potential target gene from gene databases and miRNA expression level. Antagonistic sequence of selected miRNA (antagomiR) was manufactured and applied to evaluate therapeutic potential in vitro thrombin injury model and in vivo ICH model. Their proposed target gene proteins and proteins related with cell survival pathway were studied by Western blot analysis three days after ICH induction. Results: Among 1088 miRNAs analyzed, eight miRNAs were significantly elevated and two miRNAs downregulated in hemorrhagic hemisphere. The level of the selected miRNAs was significantly upregulated in striatum after ICH and attenuating their expression by antagomiR treatment increased cell survival from in vitro thrombin injury. The antagomiR treatment improved neurological function four weeks after ICH. Inflammatory cell infiltration and apoptotic cell death were markedly reduced by antagomiR treatment, with up-regulated insulin like growth factor receptor 1 (IGF1R) protein and phosphorylated serine threonine kinase (p-Akt). Discussion: This study shows distinct miRNA expression pattern after ICH and their region specific expression. The modulation by antisense miRNA sequence reduced apoptotic Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
cell death and enhanced neurological recovery by activating IGF1R related cell survival pathway, suggesting potential therapeutic target of ICH.
599. Adeno-Associated Virus Type 6 Is Retrogradely Transported in the Non-Human Primate Brain, Implications for Gene Delivery in Huntington’s Disease
Waldy San Sebastian,1 Lluis Samaranch,1 Adrian P. Kells,1 John Bringas,1 Phillip Pivirotto,1 John Forsayeth,1 Krystof S. Bankiewicz.1 1 Department of Neurological Surgery, University of California, San Francisco, CA. Previously, we demonstrated that axonal transport of adenoassociated virus (AAV) is serotype-dependent. For instance, we have shown that AAV2 is anterogradely transported (e.g., from cell bodies to nerve terminals) in rat and non-human primate (NHP) brains but AAV6 is retrogradely transported from terminals to neuronal cells bodies in the rat brain. In the present study, we investigated axonal transport of AAV6 in NHP brain. Two cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by (magnetic resonance (MR)-guided convection-enhanced delivery. Approximately 1 month after infusion, animals were euthanized and histological analysis of AAV6-GFP distribution and tropism based on GFP expression was performed. Immunohistochemical staining of brain sections revealed GFP expression within both caudate and putamen that corresponded well with MR signal observed during gene delivery. Just as observed previously in rat brain, GFP expression was also found throughout the prefrontal, frontal, temporal and parietal cortex as well as substantia nigra pars compacta neurons, and thalamus indicating retrograde transport of the vector. AAV6-GFP preferentially transduced neurons, although few astrocytes were positive for GFP staining as well. Retrograde axonal transport of AAV-6 from single striatal infusion allows for efficient and diffused transduction of cortical neurons that otherwise would be almost impossible to achieve. AAV6 retrograde transport in the NHP brain has implications for gene therapy of neurological diseases such as Huntington’s disease where both cortical and striatal neurons are affected.
600. Herpes Simplex Virus 2 Gene, ICP10PK, Protects Against Neuronal Cell Death Associated with Valproic Acid-Induced Autism Spectrum Disorder
Dominique Bollino,1 Laure Aurelian.1 1 Pharmacology, University of Maryland, Baltimore, Baltimore, MD.
Autism spectrum disorder (ASD) is a neuro-developmental disorder of unknown etiology characterized by behavioral impairments, such as hyperactivity and impulsivity, as well as difficulties with social interactions and communication. Valproic acid (VPA), a drug used to treat epilepsy and bipolar disorder, is an environmental trigger reported to cause ASD. Recent studies indicate that VPA administered postnatally causes neurodegeneration, however, the mechanism is still poorly understood. We report that VPA (1mM) caused a time dependent increase in the death of neuronally differentiated PC12 cells, as determined by trypan blue staining. VPA-induced cell death was associated with calpain activation, as determined by the inhibition of the p28 regulatory subunit and nuclear translocation of the calpain-released apoptosis inducing factor (AIF) that causes DNA degradation. VPA did not activate caspases -3 and -7, but it induced the release of Smac/DIABLO and triggered the cleavage of receptor interacting protein-1 (RIP1), both of which can promote necroptosis, a newly defined form of programmed necrosis, and induced TNF- S229
NEUROLOGIC GENE & CELL THERAPY II expression associated with RIP-1 cleavage. VPA-induced cell death was partially inhibited by the calpain-specific inhibitor PD150606, but not by the pan caspase inhibitor z-VAD-fmk. Significant inhibition of cell death and TNF- expression was achieved through inhibition of necroptosis, using necrostatin-1, indicating that VPA induces necroptosis. Calpain inhibition restored the levels of full length RIP-1, suggesting that calpain activation contributes to VPA-induced necroptosis. Smac/DIABLO appears to contribute to the regulation of cell death through inhibition of the anti-apoptotic protein XIAP, and VPA-induced cell death was facilitated by the inhibition of the PI3K/ Akt/mTOR survival pathway, as evidenced by a significant decrease in the levels of activated (phosphorylated) Akt (pAkt) and mammalian target of Rapamycin (mTOR) (pmTOR). Interestingly, neuronally differentiated PC12 cells stably transfected with the neuroprotective herpes simplex virus gene ICP10PK were significantly protected from cell death through the inhibition of calpain, AIF, and Smac/DIABLO release as well as restored PI3K/Akt/mTOR signaling, underscoring the promising therapeutic potential of ICP10PK for ASD-associated neuronal degeneration. Future studies will determine the role of calpain activation in VPA-induced necroptosis and determine its effect on the inhibition of the PI3K/Akt/mTOR survival pathway.
601. Targeted Gene Transfer into the Ependymal Cells and Long-Term Enzyme Replacement in the CSF by Intraventricular Injection of AAV Vector Serotype 1
Yoshiyuki Yamazaki,1 Kohei Hironaka,1 Yukihiko Hirai,1 Koichi Miyake,1 Takashi Shimada.1 1 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Lysosomal storage disease (LDS) is a diverse group of genetic disorders characterized by inherited deficiency of specific lysosomal enzymes and consequently accumulation of substances to be digested within lysosomes. Systemic enzyme replacement therapy (ERT) has been successfully applied to treat certain LSDs. However, the clinical efficacy of ERT for LSD with neurological symptoms like metachromatic leukodystrophy (MLD), arylsulfatase A (ASA) deficiency, is very limited, because lysosomal enzymes cannot cross the blood-brain barrier (BBB). A possible approach to deliver the protein to the central nervous system (CNS) is direct injection of viral vector into the brain parenchyma. We have previously shown that a single injection of AAV vector into hippocampus results in the wide distribution of therapeutic protein in the mouse brain. However, to apply this approach for large animals including human, multiple vector injections with invasive surgical trepanation of the skull are required. Another approach is enzyme replacement in the cerebrospinal fluid (CSF) which enhances enzyme distribution within the CNS. Repeated infusions of recombinant protein through either intrathecal or intraventricular delivery have been shown to improve neurological symptoms in model animals with neuropathic LSDs. In this study, we tried to establish a gene therapy strategy for continuous enzyme replacement in the CSF. A preliminary experiment showed that ependymal cells and the choroid plexus in the ventricles were efficiently transduced with AAV1 but not AAV9 after intraventricular vector injection. When AAV1 vector (1x1013 vg/ml) expressing human ASA (hASA) was injected into the lateral ventricle of adult C57BL/6J mice, wide-spread transduction of ependymal cells throughout the cerebroventricular system and stable expression of hASA in those cells was observed for more than 6 months. The hASA secreted from transduced cells was detected in the CSF until 6 weeks after injection, while we could not detect hASA at 12 weeks post injection. Immunological examination showed that anti-hASA antibodies were generated in the immunocompetent C57BL/6J mice around 6 weeks post injection. In addition, when mice were tolerized by injection of purified hASA ahead of vector injection, hASA was detectable even S230
12 weeks after injection. Our results suggest that the ependymal cells can be utilized as a biological reservoir for long-term continuous secretion of therapeutic proteins in the CSF and therefore, this gene therapy protocol should be useful for treatment of LSDs with severe CNS involvement.
602. Neuroprotective Effect of a Cell-Free Extract Derived from Human Adipose Stem Cells in Experimental Stroke Models
Jung Ick Byun,1 Daejong Jeon,2 Kon Chu,1 Soon-Tae Lee,1 KeunHwa Jung,1 Jae-Jun Ban,1 Hye-Jin Yoon,1 Seungmoon Jung,2 Hyunwoo Yang,2 Byung Sun Kim,2 Ji Ye Choi,2 Jeong-Min Kim,1 Chong-Hyun Won,3 Manho Kim,1 Sang Kun Lee,1 Jae-Kyu Roh.1 1 Department of Neurology, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; 2 Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea; 3Department of Dermatology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. A recent study suggested that a cell-free extract of human adipose stem cells (hASCs-E) has beneficial effects on neurological diseases by modulating the host environment. Here, we investigated the effects of an hASCs-E in several experimental models of stroke in vitro (oxygen and glucose deprivation, OGD) and in vivo (transient or permanent focal cerebral ischemia and intracerebral hemorrhage, ICH). Ischemia was induced in vitro in Neuro2A cells, and the hASCs-E was applied 24 hour before the OGD or concurrently. Focal cerebral ischemia was induced by unilateral intraluminal thread occlusion of the middle cerebral artery (MCA) in rats for 90 minutes or permanently, or by unilateral MCA microsurgical direct electrocoagulation in mice. The ICH model was induced with an intracerebral injection of collagenase in rats. The hASCs-E was intraperitoneally administered 1 hour after the stroke insults. Neuro2a cells that had been treated with the hASCs-E showed increased viability in the WST-1 assay (P < 0.0001, Fig. 1B) and LDH assay (P < 0.001) compared to the other groups (PBS-, heattreated hASCs-E-, DNA-, RNA-, and lipid-treated cells). The rats in the permanent MCA occlusion model treated with hASCs-E 1 hour (n = 9, 174.02 ± 25.94 mm3) after occlusion showed reduced ischemic volume compared to the control rats treated with PBS (n = 11, 261.24 ± 7.03 mm3) (P < 0.05). The ICH model treated with hASCs-E exhibited better performance on the modified limb placing test. The expression of many genes related to inflammation, immune response, and cell-death were changed substantially in the ischemic rats or neuronal cells treated with the hASCs-E. These results reveal a neuroprotective role of hASCs-E in animal models of stroke, and suggest the feasible application of stem cell-based, noninvasive therapy for treating stroke.
603. Artificial MicroRNAs Against Spliced Variants of the Gene C9ORF72, the Major Cause for Familial Amyotrophic Lateral Sclerosis
Gabriela Toro,2 Florie Borel,2 Peter Sapp,1 Lina Song,2 Robert Brown,1 Christian Mueller.2 1 Neurobiology, University of Massachusetts Medical School, Worcester, MA; 2Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA. Amyotrophic Lateral Sclerosis (ALS), commonly known as Lou Gehrig’s disease is a fatal neurodegenerative disease that affects upper and lower motor neurons causing progressive muscle deterioration. The average age of onset is 60 years, and life expectancy ranges from 2 to 5 years after presenting the symptoms. Ninety-five percent of patients are diagnosed with a sporadic form of ALS as opposed to the inherited Familial ALS (FALS). There is no known cure for Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
598. The Therapeutic Potential of MicroRNA Regulation in a Rat Intracerebral Hemorrhage Model
Jangsup Moon,1 Jeong-Min Kim,1 Jung Ick Byun,1 Kon Chu,1 Keun-Hwa Jung,1 Soon-Tae Lee,1 Jin-Hee Kim,2 Dong-Kyu Park,2 So-Yoon Kim,2 Jae-Kyu Roh.1 1 Department of Neurology, Seoul National University Hospital, Seoul, Republic of Korea; 2Laboratory for Neurotherapeutics, Seoul National University College of Medicine, Seoul, Republic of Korea. Background: MicroRNAs (miRNA) are short RNA sequences which regulate gene expression by modulating post-transcriptional process, which are involved in multiple signal pathways including neuronal development and injury. Intracerebral hemorrhage is one of the most devastating diseases with significant morbidity, but treatment option is still limited. We tried to evaluate potential therapeutic target of hemorrhagic stroke by miRNA analysis. Methods: We harvested hemorrhagic brain after 24 hours from a collagenase-induced rat ICH model and extracted RNA samples from each brain hemisphere. We performed miRNAs microarray analysis to compare their expression pattern difference between left hemorrhagic hemisphere and contralateral right hemisphere, and their expression levels were confirmed by quantitative real time polymerase chain reaction. We selected miRNAs for the therapeutic target study after reviewing potential target gene from gene databases and miRNA expression level. Antagonistic sequence of selected miRNA (antagomiR) was manufactured and applied to evaluate therapeutic potential in vitro thrombin injury model and in vivo ICH model. Their proposed target gene proteins and proteins related with cell survival pathway were studied by Western blot analysis three days after ICH induction. Results: Among 1088 miRNAs analyzed, eight miRNAs were significantly elevated and two miRNAs downregulated in hemorrhagic hemisphere. The level of the selected miRNAs was significantly upregulated in striatum after ICH and attenuating their expression by antagomiR treatment increased cell survival from in vitro thrombin injury. The antagomiR treatment improved neurological function four weeks after ICH. Inflammatory cell infiltration and apoptotic cell death were markedly reduced by antagomiR treatment, with up-regulated insulin like growth factor receptor 1 (IGF1R) protein and phosphorylated serine threonine kinase (p-Akt). Discussion: This study shows distinct miRNA expression pattern after ICH and their region specific expression. The modulation by antisense miRNA sequence reduced apoptotic Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
cell death and enhanced neurological recovery by activating IGF1R related cell survival pathway, suggesting potential therapeutic target of ICH.
599. Adeno-Associated Virus Type 6 Is Retrogradely Transported in the Non-Human Primate Brain, Implications for Gene Delivery in Huntington’s Disease
Waldy San Sebastian,1 Lluis Samaranch,1 Adrian P. Kells,1 John Bringas,1 Phillip Pivirotto,1 John Forsayeth,1 Krystof S. Bankiewicz.1 1 Department of Neurological Surgery, University of California, San Francisco, CA. Previously, we demonstrated that axonal transport of adenoassociated virus (AAV) is serotype-dependent. For instance, we have shown that AAV2 is anterogradely transported (e.g., from cell bodies to nerve terminals) in rat and non-human primate (NHP) brains but AAV6 is retrogradely transported from terminals to neuronal cells bodies in the rat brain. In the present study, we investigated axonal transport of AAV6 in NHP brain. Two cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by (magnetic resonance (MR)-guided convection-enhanced delivery. Approximately 1 month after infusion, animals were euthanized and histological analysis of AAV6-GFP distribution and tropism based on GFP expression was performed. Immunohistochemical staining of brain sections revealed GFP expression within both caudate and putamen that corresponded well with MR signal observed during gene delivery. Just as observed previously in rat brain, GFP expression was also found throughout the prefrontal, frontal, temporal and parietal cortex as well as substantia nigra pars compacta neurons, and thalamus indicating retrograde transport of the vector. AAV6-GFP preferentially transduced neurons, although few astrocytes were positive for GFP staining as well. Retrograde axonal transport of AAV-6 from single striatal infusion allows for efficient and diffused transduction of cortical neurons that otherwise would be almost impossible to achieve. AAV6 retrograde transport in the NHP brain has implications for gene therapy of neurological diseases such as Huntington’s disease where both cortical and striatal neurons are affected.
600. Herpes Simplex Virus 2 Gene, ICP10PK, Protects Against Neuronal Cell Death Associated with Valproic Acid-Induced Autism Spectrum Disorder
Dominique Bollino,1 Laure Aurelian.1 1 Pharmacology, University of Maryland, Baltimore, Baltimore, MD.
Autism spectrum disorder (ASD) is a neuro-developmental disorder of unknown etiology characterized by behavioral impairments, such as hyperactivity and impulsivity, as well as difficulties with social interactions and communication. Valproic acid (VPA), a drug used to treat epilepsy and bipolar disorder, is an environmental trigger reported to cause ASD. Recent studies indicate that VPA administered postnatally causes neurodegeneration, however, the mechanism is still poorly understood. We report that VPA (1mM) caused a time dependent increase in the death of neuronally differentiated PC12 cells, as determined by trypan blue staining. VPA-induced cell death was associated with calpain activation, as determined by the inhibition of the p28 regulatory subunit and nuclear translocation of the calpain-released apoptosis inducing factor (AIF) that causes DNA degradation. VPA did not activate caspases -3 and -7, but it induced the release of Smac/DIABLO and triggered the cleavage of receptor interacting protein-1 (RIP1), both of which can promote necroptosis, a newly defined form of programmed necrosis, and induced TNF- S229
NEUROLOGIC GENE & CELL THERAPY II expression associated with RIP-1 cleavage. VPA-induced cell death was partially inhibited by the calpain-specific inhibitor PD150606, but not by the pan caspase inhibitor z-VAD-fmk. Significant inhibition of cell death and TNF- expression was achieved through inhibition of necroptosis, using necrostatin-1, indicating that VPA induces necroptosis. Calpain inhibition restored the levels of full length RIP-1, suggesting that calpain activation contributes to VPA-induced necroptosis. Smac/DIABLO appears to contribute to the regulation of cell death through inhibition of the anti-apoptotic protein XIAP, and VPA-induced cell death was facilitated by the inhibition of the PI3K/ Akt/mTOR survival pathway, as evidenced by a significant decrease in the levels of activated (phosphorylated) Akt (pAkt) and mammalian target of Rapamycin (mTOR) (pmTOR). Interestingly, neuronally differentiated PC12 cells stably transfected with the neuroprotective herpes simplex virus gene ICP10PK were significantly protected from cell death through the inhibition of calpain, AIF, and Smac/DIABLO release as well as restored PI3K/Akt/mTOR signaling, underscoring the promising therapeutic potential of ICP10PK for ASD-associated neuronal degeneration. Future studies will determine the role of calpain activation in VPA-induced necroptosis and determine its effect on the inhibition of the PI3K/Akt/mTOR survival pathway.
601. Targeted Gene Transfer into the Ependymal Cells and Long-Term Enzyme Replacement in the CSF by Intraventricular Injection of AAV Vector Serotype 1
Yoshiyuki Yamazaki,1 Kohei Hironaka,1 Yukihiko Hirai,1 Koichi Miyake,1 Takashi Shimada.1 1 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Lysosomal storage disease (LDS) is a diverse group of genetic disorders characterized by inherited deficiency of specific lysosomal enzymes and consequently accumulation of substances to be digested within lysosomes. Systemic enzyme replacement therapy (ERT) has been successfully applied to treat certain LSDs. However, the clinical efficacy of ERT for LSD with neurological symptoms like metachromatic leukodystrophy (MLD), arylsulfatase A (ASA) deficiency, is very limited, because lysosomal enzymes cannot cross the blood-brain barrier (BBB). A possible approach to deliver the protein to the central nervous system (CNS) is direct injection of viral vector into the brain parenchyma. We have previously shown that a single injection of AAV vector into hippocampus results in the wide distribution of therapeutic protein in the mouse brain. However, to apply this approach for large animals including human, multiple vector injections with invasive surgical trepanation of the skull are required. Another approach is enzyme replacement in the cerebrospinal fluid (CSF) which enhances enzyme distribution within the CNS. Repeated infusions of recombinant protein through either intrathecal or intraventricular delivery have been shown to improve neurological symptoms in model animals with neuropathic LSDs. In this study, we tried to establish a gene therapy strategy for continuous enzyme replacement in the CSF. A preliminary experiment showed that ependymal cells and the choroid plexus in the ventricles were efficiently transduced with AAV1 but not AAV9 after intraventricular vector injection. When AAV1 vector (1x1013 vg/ml) expressing human ASA (hASA) was injected into the lateral ventricle of adult C57BL/6J mice, wide-spread transduction of ependymal cells throughout the cerebroventricular system and stable expression of hASA in those cells was observed for more than 6 months. The hASA secreted from transduced cells was detected in the CSF until 6 weeks after injection, while we could not detect hASA at 12 weeks post injection. Immunological examination showed that anti-hASA antibodies were generated in the immunocompetent C57BL/6J mice around 6 weeks post injection. In addition, when mice were tolerized by injection of purified hASA ahead of vector injection, hASA was detectable even S230
12 weeks after injection. Our results suggest that the ependymal cells can be utilized as a biological reservoir for long-term continuous secretion of therapeutic proteins in the CSF and therefore, this gene therapy protocol should be useful for treatment of LSDs with severe CNS involvement.
602. Neuroprotective Effect of a Cell-Free Extract Derived from Human Adipose Stem Cells in Experimental Stroke Models
Jung Ick Byun,1 Daejong Jeon,2 Kon Chu,1 Soon-Tae Lee,1 KeunHwa Jung,1 Jae-Jun Ban,1 Hye-Jin Yoon,1 Seungmoon Jung,2 Hyunwoo Yang,2 Byung Sun Kim,2 Ji Ye Choi,2 Jeong-Min Kim,1 Chong-Hyun Won,3 Manho Kim,1 Sang Kun Lee,1 Jae-Kyu Roh.1 1 Department of Neurology, Biomedical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea; 2 Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea; 3Department of Dermatology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea. A recent study suggested that a cell-free extract of human adipose stem cells (hASCs-E) has beneficial effects on neurological diseases by modulating the host environment. Here, we investigated the effects of an hASCs-E in several experimental models of stroke in vitro (oxygen and glucose deprivation, OGD) and in vivo (transient or permanent focal cerebral ischemia and intracerebral hemorrhage, ICH). Ischemia was induced in vitro in Neuro2A cells, and the hASCs-E was applied 24 hour before the OGD or concurrently. Focal cerebral ischemia was induced by unilateral intraluminal thread occlusion of the middle cerebral artery (MCA) in rats for 90 minutes or permanently, or by unilateral MCA microsurgical direct electrocoagulation in mice. The ICH model was induced with an intracerebral injection of collagenase in rats. The hASCs-E was intraperitoneally administered 1 hour after the stroke insults. Neuro2a cells that had been treated with the hASCs-E showed increased viability in the WST-1 assay (P < 0.0001, Fig. 1B) and LDH assay (P < 0.001) compared to the other groups (PBS-, heattreated hASCs-E-, DNA-, RNA-, and lipid-treated cells). The rats in the permanent MCA occlusion model treated with hASCs-E 1 hour (n = 9, 174.02 ± 25.94 mm3) after occlusion showed reduced ischemic volume compared to the control rats treated with PBS (n = 11, 261.24 ± 7.03 mm3) (P < 0.05). The ICH model treated with hASCs-E exhibited better performance on the modified limb placing test. The expression of many genes related to inflammation, immune response, and cell-death were changed substantially in the ischemic rats or neuronal cells treated with the hASCs-E. These results reveal a neuroprotective role of hASCs-E in animal models of stroke, and suggest the feasible application of stem cell-based, noninvasive therapy for treating stroke.
603. Artificial MicroRNAs Against Spliced Variants of the Gene C9ORF72, the Major Cause for Familial Amyotrophic Lateral Sclerosis
Gabriela Toro,2 Florie Borel,2 Peter Sapp,1 Lina Song,2 Robert Brown,1 Christian Mueller.2 1 Neurobiology, University of Massachusetts Medical School, Worcester, MA; 2Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA. Amyotrophic Lateral Sclerosis (ALS), commonly known as Lou Gehrig’s disease is a fatal neurodegenerative disease that affects upper and lower motor neurons causing progressive muscle deterioration. The average age of onset is 60 years, and life expectancy ranges from 2 to 5 years after presenting the symptoms. Ninety-five percent of patients are diagnosed with a sporadic form of ALS as opposed to the inherited Familial ALS (FALS). There is no known cure for Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy