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240. Nicotinic alpha7 Receptor Gene Transfer into Brain
GENE THERAPY FOR THE NERVOUS SYSTEM I rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO ) by the monofilament method. Animals were treated with intravenricular administration of 50μl of adenoviral suspension (containing2×108p.f.u./50μl ) 2 hours after the onset of MCAO (on reperfusion). Neurological functional tests (neurological severity score) were performed before and on day 1,4,7,14,21,28,and 35 after MCAO. We found that intravenricular administration of FGF2-expressing adeoviral vectors markedly enhanced recovery of neurological severity score (p<0.05 )during the first month after stroke without apparent adverse effects. In addition, we confirmed that postischemic administration of this vector induced a level of neuroprotection satisfactory for infarct size reduction. These results suggest that FGF2 gene transfer using these adenoviral vectors might be useful for the treatment of occlusive cerebrovascular disease.
240. Nicotinic alpha7 Receptor Gene Transfer into Brain Ke Ren,1 Thaddeus Papke,2 Jeffry A. Hughes,1 Roger L. Papke,2 Vladimir Uteshev,2 Clare Stokes,2 Edwin M. Meyer.2 1 Department of Pharmaceutics, University of Florida, Gainesville, FL, United States; 2Department of Pharmacology, University of Florida, Gainesville, FL, United States. Brain nicotinic alpha7 receptors are implicated in Alzheimer’s disease through their actions on memory related behaviors, binding to beta-amyloid, and up-regulation in transgenic animals overexpressing these peptides. Pharmacological manipulation of these receptors appears to protect neurons from amyloid-exposure and other models associated with Alzheimer’s disease. However, these receptors rapidly desensitize, complicating the effects of agonists in vivo. Another approach to investigate the effects of alpha7 receptors on Alzheimer’s disease-related processes involves elevating the levels of this receptor in the plasma membrane. We therefore took a somatic gene transfer approach to modify the expression of alpha7 receptors in the adult brain, using an rAAV2 vector system found previously to be effective in brain neurons. This rAAV2-ra7 vector was constructed to contain the CBA promoter/enhancer and downstream WPRE sequence. Rat alpha7 cDNA was subcloned into this plasmid using Pst1 and Cla1, and directionality confirmed by full length sequence analyses. The biological activity of the sense orientation was confirmed by calcium phosphate transfection of rat GH4C1 cells, one of the very few lines the permits functional alpha7 expression and membrane incorporation. Functional alpha7 receptor binding was demonstrated with high affinity, nicotine (1 mM)displaceable, methyllycacotinine (MLA) binding. Cells transfected with the same plasmid, except expressing GFP instead of alpha7, had no MLA binding. The plasmid AAV2-ra7 transfected cells expressed increasingly high levels of MLA binding between 0-5 days, with the highest level seen (109 ± 8 pmol/mg protein, mean ± SEM, N = 3) much higher than in rat PC12 cells (24 ± 1 pmol/mg protein), a model system for studying this receptor. The rAAV2-ra7 vector was encapsulated as described previously in our lab and injected into two brain regions: striatum (3 ul, 3 x 105 genomic particles) which normally expresses very low levels of the receptor, and hippocampus (2 x 2 ul, 4 x 105 particles), where the receptor is found in high density and is important for memory related behaviors. These low vector doses were selected in initial studies to avoid potential toxicities associated with this highly calcium-permeant receptor, comparable to the NMDA R1 receptor. Non-quantitative RT-PCR revealed alpha7 mRNA in both regions after transduction. High affinity MLA binding increased in the hippocampus after transduction with the rAAV2-ra7 (143 ± 13% of contralateral control binding, mean ± SEM). However, no increase in alpha7 receptor density was observed in striatum. These results support the recent hypothesis that functional alpha7 receptor integration into plasma membranes requires additional cofactors intrinsic to alpha7 – Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
expressing neurons. Confocal microscopy using fluorescent-labelled alpha-bungarotoxin (BTX) binding demonstrated no change in striatal receptor density or distribution but did appear to increase hippocampal hilar membrane-bound receptor density without affecting distribution. These results suggest the utility of rAAV2 gene transfer for increasing membrane alpha7 nicotinic receptor density predominantly in selected brain neurons.
241. Effects of the Sustained Expression of GDNF in Fetal Dopaminergic Grafts Enni Lehtonen,1,2 David Blum,1,2 Olivier Bockstael,1,2 Abdelwahed Chtarto,1,2 Catherine Melas,1,2 Marc Peschanski,3 Thierry Velu,1 Jacques Brotchi,2 Marc Levivier,2 Liliane Tenenbaum.1,2 1 Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Free University of Brussels, Brussels, Belgium; 2 Laboratory of Experimental Neurosurgery, Free University of Brussels, Brussels, Belgium; 3Unité 421, INSERM, Créteil, France. Rationale: We have previously shown that AAV2 vectors efficiently transduce E14 rat ventral mesencephalic (VM) transplants (Lehtonen et al., 2002). Continuous release of GDNF by VM grafts could i) enhance the reinnervation potential of grafted dopaminergic neurons; ii) protect remaining host dopaminergic neurons against ongoing degeneration. Experiment: To evaluate the effect of virally-expressed GDNF inside the graft on reinnervation, E14 fetal mesencephalon was genetically-modified using an AAV vector expressing GDNF or EGFP under the control of a CMV promoter, prior to transplantation into the striatum of rats with a complete 6-hydroxydopamine lesion. Results: The number of dopaminergic (tyrosine hydroxylase-positive) neurons was 2-fold higher in the GDNF group as compared to the EGFP group. Reinnervation of the striatum was also significantly enhanced in animals with GDNF-secreting grafts. No effect on the rotational behaviour was observed. Perspectives: In the future, the protective effect of GDNF secreted by the graft on dopaminergic neurons of the host substantia nigra will be evaluated in a progressive model of Parkinson’s disease. E.Lehtonen, F.Bonnaud, C.Melas, A.Lubansu, B.Malgrange, A.Chtarto, T. Velu, J.Brotchi , M. Peschanski, M. Levivier and L.Tenenbaum. AAV vectors mediate efficient and sustained transduction of rat embryonic mesencephalon. NeuroReport, 13: 1503-1507; 2002.
242. Characterization of a Huntingtin-Specific Single-Chain Fv Intrabody in a Huntington’s Disease Striatal Cell Model Yangsheng Zhou,1,2 Todd W. Miller,1,2 Thomas L. Shirley,1,2 Anne Messer.1,2 1 Division of Genetic Disorders, Wadsworth Center, NYS DOH, Albany, NY; 2Department of Biomedical Sciences, University at Albany, Albany, NY. Huntington’s disease (HD) is a member of a family of neurodegenerative disorders caused by expanded CAG repeats encoding polyglutamine. The extended polyglutamine causes the huntingtin protein to form insoluble aggregates and engage in aberrant protein-protein interactions. Therapeutic intervention to prevent aggregation, reduce abnormal interactions, and allow proper proteolytic processing may reduce HD pathogenesis. Intrabodies (single-chain Fv antibody fragments) can bind with high specificity S95
240. Nicotinic alpha7 Receptor Gene Transfer into Brain Ke Ren,1 Thaddeus Papke,2 Jeffry A. Hughes,1 Roger L. Papke,2 Vladimir Uteshev,2 Clare Stokes,2 Edwin M. Meyer.2 1 Department of Pharmaceutics, University of Florida, Gainesville, FL, United States; 2Department of Pharmacology, University of Florida, Gainesville, FL, United States. Brain nicotinic alpha7 receptors are implicated in Alzheimer’s disease through their actions on memory related behaviors, binding to beta-amyloid, and up-regulation in transgenic animals overexpressing these peptides. Pharmacological manipulation of these receptors appears to protect neurons from amyloid-exposure and other models associated with Alzheimer’s disease. However, these receptors rapidly desensitize, complicating the effects of agonists in vivo. Another approach to investigate the effects of alpha7 receptors on Alzheimer’s disease-related processes involves elevating the levels of this receptor in the plasma membrane. We therefore took a somatic gene transfer approach to modify the expression of alpha7 receptors in the adult brain, using an rAAV2 vector system found previously to be effective in brain neurons. This rAAV2-ra7 vector was constructed to contain the CBA promoter/enhancer and downstream WPRE sequence. Rat alpha7 cDNA was subcloned into this plasmid using Pst1 and Cla1, and directionality confirmed by full length sequence analyses. The biological activity of the sense orientation was confirmed by calcium phosphate transfection of rat GH4C1 cells, one of the very few lines the permits functional alpha7 expression and membrane incorporation. Functional alpha7 receptor binding was demonstrated with high affinity, nicotine (1 mM)displaceable, methyllycacotinine (MLA) binding. Cells transfected with the same plasmid, except expressing GFP instead of alpha7, had no MLA binding. The plasmid AAV2-ra7 transfected cells expressed increasingly high levels of MLA binding between 0-5 days, with the highest level seen (109 ± 8 pmol/mg protein, mean ± SEM, N = 3) much higher than in rat PC12 cells (24 ± 1 pmol/mg protein), a model system for studying this receptor. The rAAV2-ra7 vector was encapsulated as described previously in our lab and injected into two brain regions: striatum (3 ul, 3 x 105 genomic particles) which normally expresses very low levels of the receptor, and hippocampus (2 x 2 ul, 4 x 105 particles), where the receptor is found in high density and is important for memory related behaviors. These low vector doses were selected in initial studies to avoid potential toxicities associated with this highly calcium-permeant receptor, comparable to the NMDA R1 receptor. Non-quantitative RT-PCR revealed alpha7 mRNA in both regions after transduction. High affinity MLA binding increased in the hippocampus after transduction with the rAAV2-ra7 (143 ± 13% of contralateral control binding, mean ± SEM). However, no increase in alpha7 receptor density was observed in striatum. These results support the recent hypothesis that functional alpha7 receptor integration into plasma membranes requires additional cofactors intrinsic to alpha7 – Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
expressing neurons. Confocal microscopy using fluorescent-labelled alpha-bungarotoxin (BTX) binding demonstrated no change in striatal receptor density or distribution but did appear to increase hippocampal hilar membrane-bound receptor density without affecting distribution. These results suggest the utility of rAAV2 gene transfer for increasing membrane alpha7 nicotinic receptor density predominantly in selected brain neurons.
241. Effects of the Sustained Expression of GDNF in Fetal Dopaminergic Grafts Enni Lehtonen,1,2 David Blum,1,2 Olivier Bockstael,1,2 Abdelwahed Chtarto,1,2 Catherine Melas,1,2 Marc Peschanski,3 Thierry Velu,1 Jacques Brotchi,2 Marc Levivier,2 Liliane Tenenbaum.1,2 1 Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Free University of Brussels, Brussels, Belgium; 2 Laboratory of Experimental Neurosurgery, Free University of Brussels, Brussels, Belgium; 3Unité 421, INSERM, Créteil, France. Rationale: We have previously shown that AAV2 vectors efficiently transduce E14 rat ventral mesencephalic (VM) transplants (Lehtonen et al., 2002). Continuous release of GDNF by VM grafts could i) enhance the reinnervation potential of grafted dopaminergic neurons; ii) protect remaining host dopaminergic neurons against ongoing degeneration. Experiment: To evaluate the effect of virally-expressed GDNF inside the graft on reinnervation, E14 fetal mesencephalon was genetically-modified using an AAV vector expressing GDNF or EGFP under the control of a CMV promoter, prior to transplantation into the striatum of rats with a complete 6-hydroxydopamine lesion. Results: The number of dopaminergic (tyrosine hydroxylase-positive) neurons was 2-fold higher in the GDNF group as compared to the EGFP group. Reinnervation of the striatum was also significantly enhanced in animals with GDNF-secreting grafts. No effect on the rotational behaviour was observed. Perspectives: In the future, the protective effect of GDNF secreted by the graft on dopaminergic neurons of the host substantia nigra will be evaluated in a progressive model of Parkinson’s disease. E.Lehtonen, F.Bonnaud, C.Melas, A.Lubansu, B.Malgrange, A.Chtarto, T. Velu, J.Brotchi , M. Peschanski, M. Levivier and L.Tenenbaum. AAV vectors mediate efficient and sustained transduction of rat embryonic mesencephalon. NeuroReport, 13: 1503-1507; 2002.
242. Characterization of a Huntingtin-Specific Single-Chain Fv Intrabody in a Huntington’s Disease Striatal Cell Model Yangsheng Zhou,1,2 Todd W. Miller,1,2 Thomas L. Shirley,1,2 Anne Messer.1,2 1 Division of Genetic Disorders, Wadsworth Center, NYS DOH, Albany, NY; 2Department of Biomedical Sciences, University at Albany, Albany, NY. Huntington’s disease (HD) is a member of a family of neurodegenerative disorders caused by expanded CAG repeats encoding polyglutamine. The extended polyglutamine causes the huntingtin protein to form insoluble aggregates and engage in aberrant protein-protein interactions. Therapeutic intervention to prevent aggregation, reduce abnormal interactions, and allow proper proteolytic processing may reduce HD pathogenesis. Intrabodies (single-chain Fv antibody fragments) can bind with high specificity S95