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205. CNS Expression of Glucocerebrosidase Corrects Hippocampal alpha-Synuclein Aggregates and Memory Deficits in a Mouse Model of Gaucher-Related Parkinsonism
NEUROLOGIC & OPHTHALMIC GENE & CELL THERAPY I is to treat ADRP by an RNA suppression and replacement approach using siRNA combined with normal, but resistant, rhodopsin gene. We tested gene therapy in a mouse model of ADRP that expresses the human P23H RHO transgene and a single copy of the mouse rhodopsin gene. We constructed a combination AAV vector containing an siRNA that degrades both mouse and human opsin mRNA and a “hardened” RHO gene (RHO301) that is resistant to that siRNA. The gene RHO301 was generated by introducing silent mutations to eliminate the siRNA cleavage site. The combination cassette (RHO301-siRNA301) or RHO-only cassette (RHO301) in AAV5 was delivered into the right eyes of transgenic mice by subretinal injection, and AAV-GFP was injected by the same method in the left eyes. Following delivery of RHO301-siRNA301 in AAV5 to P23H RHO transgenic mice, the electroretinogram (ERG) a-wave and b-wave response was significantly increased in treated eyes relative to control treated eyes for more than 3 months, suggesting preservation of retinal function. To assess retinal structure, the outer nuclear layer (ONL) thickness was measured with the optical coherence tomography (OCT) in living mice at 3 months post injection. The integrity of treated eyes was significantly greater compared with of control treated eyes. Augmentation by delivery of RHO301 alone did not show consistent improvement in ERG response over the same time course in the same mouse model. AAV delivery of a combination construct containing gene encoding wild-type rhodopsin and RHOspecific siRNA improved retinal function and structure in this ADRP model, suggesting that this approach may be useful for the treatment of this inherited blinding disorder.
205. CNS Expression of Glucocerebrosidase Corrects Hippocampal alpha-Synuclein Aggregates and Memory Deficits in a Mouse Model of Gaucher-Related Parkinsonism Pablo S. Sardi,1 Lamya S. Shihabuddin,1 Seng H. Cheng.1 Genzyme Corporation, Framingham, MA.
1
Emerging genetic and clinical evidence suggest a link between Gaucher disease and the synucleinopathies, Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Gaucher patients harboring mutations in the glucocerebrosidase gene (GBA) present an increased risk for Parkinsonism and subjects with sporadic PD and DLB exhibit an increased occurrence of mutations in GBA. Lewy bodylike alpha-synuclein inclusions, a neuropathological hallmark of PD, have also been found in patients with GD. To better understand the relationship between mutant GBA and alpha-synuclein accumulation, the behavior and brain histopathology of two mouse models of GD (GbaD409v/D409V and Gba-/+) were examined. Both mouse models of Gaucher disease exhibited features of synucleinopathies such as the progressive accumulation of proteinase K-resistant alpha-synuclein/ ubiquitin aggregates in hippocampal neurons. Co-incident with the appearance of this pathology was a loss in memory as determined using novel object recognition and contextual fear conditioning tests. Hippocampal aggregates of alpha-synuclein/ubiquitin were apparent in Gaucher mice with less than 60% glucocerebrosidase activity provided there was concurrent presence of a mutant Gba allele. This suggests that the presence of mutant glucocerebrosidase might confer a gain in toxic function. However, although the presence of aggregates was necessary, it was not sufficient to engender the observed impairment in hippocampal memory. Importantly, AAV1-mediated expression of glucocerebrosidase in the CNS of GbaD409v/D409V mice ameliorated both the pathological accumulation of alpha-synuclein/ ubiquitin aggregates and memory deficit. Together, these data support the contention that mutations in Gba can render the brains of mice with Parkinsonism-like pathology and that reconstituting the CNS with exogenous glucocerebrosidase activity may represent a potential therapeutic strategy for Gba-associated synucleinopathies. S80
206. Anterograde Trafficking of AAV2 Vectors in the Primate Brain
Adrian P. Kells,1 Ernesto Aguilar Salegio,1 Krystof S. Bankiewicz.1 1 Neurosurgery, University of California San Francisco, San Francisco, CA. Neuro gene delivery via direct intrapranchymal injections of adenoassociated viral (AAV) vectors is a locally administered treatment that requires accurate delivery to maximize safety and efficacy. The large volume and convoluted architecture of the human brain is a barrier to translating small animal findings into clinical procedures. Too little target coverage and the treatment risks being ineffective. Excessive distribution or off-target delivery raises the possibility of adverse effects. In order to target large structures such as the putamen for the treatment of Parkinson’s disease (PD), we have developed real-time MRI monitored convection-enhanced delivery (CED) demonstrating safe and reliable delivery of large volume (>300µL) AAV vector infusions in non-human primates (NHP). Subsequent to our discovery that AAV2 vectors undergo anterograde transport along thalamocortical projections, we have now analyzed prior NHP studies to look for evidence of anterograde transport of AAV2 in other brain regions. We have conducted multiple NHP studies to investigate the delivery of AAV2-GDNF (glial-derived neurotrophic factor) to the putamen or substantia nigra as a neuro-regenerative treatment for PD. These studies have shown delivery of AAV2-GDNF into NHP putamen is well tolerated with significant functional improvements and restoration of dopaminergic activity, however delivery to the substantia nigra resulted in weight loss. Additional assessment of these NHP studies shows anterograde distribution of AAV2-GDNF vector resulting in the transduction of neurons and expression of GDNF in secondary regions of the brain not directly infused. Of particular interest is the equivalent transduction of nigral neurons after putamen delivery in both MPTP-lesioned and naïve NHP indicating that GDNF expression in nigra is not dependent on the integrity of the dopaminergic neurons. Double staining with tyrosine hydroxylase (TH) showed that almost all GDNF in the substantia nigra was localized to TH-negative fibers and cell bodies within the pars reticulata. GDNF expression in the globus pallidus and sub-thalamic nucleus is further evidence of anterograde transport with no GDNF observed in non-basal ganglia nuclei. Conversely, AAV2-GDNF delivery to the substantia nigra resulted in significant co-localization of GDNF with TH-positive pars compacta neurons, however transduction was not limited to the nigra but included the hypothalamus, sub thalamic nucleus, globus pallidus, thalamus, septum and caudate nucleus. This GDNF presence in non-dopaminergic areas is indicative of protein and vector trafficking that may have contributed to the weight loss and demonstrates the non-localized gene delivery that can occur with AAV2. We have confirmed anterograde transport of AAV2 by PCR detection of vector DNA in non-infused regions of an AAV2-GFP treated NHP brain. In conclusion, AAV2 gene delivery is not localized to the infusion site and consideration should be given to the potential effects of transgene expression in anterogrdely connected areas from primary target brain region. Furthermore, we believe that anterograde axonal trafficking pattern of AAV2 will occur in other brain regions outside of the thalamocortical and basal ganglia circuits.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
205. CNS Expression of Glucocerebrosidase Corrects Hippocampal alpha-Synuclein Aggregates and Memory Deficits in a Mouse Model of Gaucher-Related Parkinsonism Pablo S. Sardi,1 Lamya S. Shihabuddin,1 Seng H. Cheng.1 Genzyme Corporation, Framingham, MA.
1
Emerging genetic and clinical evidence suggest a link between Gaucher disease and the synucleinopathies, Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Gaucher patients harboring mutations in the glucocerebrosidase gene (GBA) present an increased risk for Parkinsonism and subjects with sporadic PD and DLB exhibit an increased occurrence of mutations in GBA. Lewy bodylike alpha-synuclein inclusions, a neuropathological hallmark of PD, have also been found in patients with GD. To better understand the relationship between mutant GBA and alpha-synuclein accumulation, the behavior and brain histopathology of two mouse models of GD (GbaD409v/D409V and Gba-/+) were examined. Both mouse models of Gaucher disease exhibited features of synucleinopathies such as the progressive accumulation of proteinase K-resistant alpha-synuclein/ ubiquitin aggregates in hippocampal neurons. Co-incident with the appearance of this pathology was a loss in memory as determined using novel object recognition and contextual fear conditioning tests. Hippocampal aggregates of alpha-synuclein/ubiquitin were apparent in Gaucher mice with less than 60% glucocerebrosidase activity provided there was concurrent presence of a mutant Gba allele. This suggests that the presence of mutant glucocerebrosidase might confer a gain in toxic function. However, although the presence of aggregates was necessary, it was not sufficient to engender the observed impairment in hippocampal memory. Importantly, AAV1-mediated expression of glucocerebrosidase in the CNS of GbaD409v/D409V mice ameliorated both the pathological accumulation of alpha-synuclein/ ubiquitin aggregates and memory deficit. Together, these data support the contention that mutations in Gba can render the brains of mice with Parkinsonism-like pathology and that reconstituting the CNS with exogenous glucocerebrosidase activity may represent a potential therapeutic strategy for Gba-associated synucleinopathies. S80
206. Anterograde Trafficking of AAV2 Vectors in the Primate Brain
Adrian P. Kells,1 Ernesto Aguilar Salegio,1 Krystof S. Bankiewicz.1 1 Neurosurgery, University of California San Francisco, San Francisco, CA. Neuro gene delivery via direct intrapranchymal injections of adenoassociated viral (AAV) vectors is a locally administered treatment that requires accurate delivery to maximize safety and efficacy. The large volume and convoluted architecture of the human brain is a barrier to translating small animal findings into clinical procedures. Too little target coverage and the treatment risks being ineffective. Excessive distribution or off-target delivery raises the possibility of adverse effects. In order to target large structures such as the putamen for the treatment of Parkinson’s disease (PD), we have developed real-time MRI monitored convection-enhanced delivery (CED) demonstrating safe and reliable delivery of large volume (>300µL) AAV vector infusions in non-human primates (NHP). Subsequent to our discovery that AAV2 vectors undergo anterograde transport along thalamocortical projections, we have now analyzed prior NHP studies to look for evidence of anterograde transport of AAV2 in other brain regions. We have conducted multiple NHP studies to investigate the delivery of AAV2-GDNF (glial-derived neurotrophic factor) to the putamen or substantia nigra as a neuro-regenerative treatment for PD. These studies have shown delivery of AAV2-GDNF into NHP putamen is well tolerated with significant functional improvements and restoration of dopaminergic activity, however delivery to the substantia nigra resulted in weight loss. Additional assessment of these NHP studies shows anterograde distribution of AAV2-GDNF vector resulting in the transduction of neurons and expression of GDNF in secondary regions of the brain not directly infused. Of particular interest is the equivalent transduction of nigral neurons after putamen delivery in both MPTP-lesioned and naïve NHP indicating that GDNF expression in nigra is not dependent on the integrity of the dopaminergic neurons. Double staining with tyrosine hydroxylase (TH) showed that almost all GDNF in the substantia nigra was localized to TH-negative fibers and cell bodies within the pars reticulata. GDNF expression in the globus pallidus and sub-thalamic nucleus is further evidence of anterograde transport with no GDNF observed in non-basal ganglia nuclei. Conversely, AAV2-GDNF delivery to the substantia nigra resulted in significant co-localization of GDNF with TH-positive pars compacta neurons, however transduction was not limited to the nigra but included the hypothalamus, sub thalamic nucleus, globus pallidus, thalamus, septum and caudate nucleus. This GDNF presence in non-dopaminergic areas is indicative of protein and vector trafficking that may have contributed to the weight loss and demonstrates the non-localized gene delivery that can occur with AAV2. We have confirmed anterograde transport of AAV2 by PCR detection of vector DNA in non-infused regions of an AAV2-GFP treated NHP brain. In conclusion, AAV2 gene delivery is not localized to the infusion site and consideration should be given to the potential effects of transgene expression in anterogrdely connected areas from primary target brain region. Furthermore, we believe that anterograde axonal trafficking pattern of AAV2 will occur in other brain regions outside of the thalamocortical and basal ganglia circuits.
Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy