- Email: [email protected]
246. Efficient Gene Transfer to Peripheral Nervous System Via Systemic Delivery of AAV Vectors
NEUROLOGIC: THERAPEUTIC APPLICATIONS temporal and spatial progression of early AD pathogenic events and potentially leading to the development of new anti-Aβ therapeutics. Supported by NIH R01AG020204 to HJF and NIH R01AG023593 to WJB.
248. Revealing the Role(s) of TNF-alpha in Early Alzheimer’s Disease Via rAAV-Mediated Hippocampal Gene Transfer in a Triple Transgenic Mouse Model Michelle C. Janelsins,2,4 Michael A. Mastrangelo,4 Keigan M. Park,3,4 Kelly L. Fowlkes,4 Maria Kinsky,4 Wade C. Narrow,4 Salvatore Oddo,5 Frank M. LaFerla,5 Howard J. Federoff,1,2,4 William J. Bowers.1,4 1 Neurology, Univ. Rochester School of Medicine and Dentistry, Rochester, NY; 2Microbiology and Immunology, Univ. Rochester School of Medicine and Dentistry, Rochester, NY; 3Pharmacology and Physiology, Univ. Rochester School of Medicine and Dentistry, Rochester, NY; 4Ctr. for Aging and Devel. Biol., Univ. Rochester School of Medicine and Dentistry, Rochester, NY; 5Neurobiology and Behavior, Univ. California-Irvine, Irvine, CA. Alzheimer’s disease is a complex neurodegenerative disorder that drastically impairs intellectual and emotional functioning in afflicted individuals. The disease is characterized pathologically by a temporal and spatial progression of amyloid-beta (Aβ) deposition, neurofibrillary tangle formation, and synaptic degeneration. Inflammatory mediators have long been proposed as being integral for initiating and/or propagating AD-associated pathologic processes within the brain. To this end, we assessed the expression of proinflammatory molecules in the recently described triple transgenic mouse model (3xTg-AD), which exhibits Aβ deposition, neurofibrillary tangle formation, and synaptic dysfunction with regional specificity similar to human AD. We observed significant up-regulation of the pro-inflammatory cytokine, TNF-α, specifically within one of the earliest affected brain regions in AD, the entorhinal cortex, prior to the onset of overt amyloid pathology. Moreover, the 3xTg-AD entorhinal cortex, unlike the hippocampus, harbored an increased number of microglia/macrophages as determined by quantification of F4/80-positive cells, further implicating the entorhinal cortex in inflammatory processes at early stages of the disease. A recombinant adeno-associated virus (rAAV) vector expressing TNF-α to create a sustained focal inflammatory response was stereotactically delivered to 3xTg-AD mice prior to the appearance of pathological signatures. In ongoing studies, we have observed significant alterations in downstream TNF-α related signaling molecule expression in transduced brain tissues and concomitant modulation of memory/learning behaviors. This integrated biochemical, immunohistochemical, and behavioral approach will provide major insights into the involvement of TNFα mediated inflammation in the temporal and spatial progression of early AD pathogenic events and carries the potential to reveal new therapeutic targets. Supported by NIH F31NS049995 to MCJ, NIH R01AG020204 to HJF, and NIH R01AG023593 to WJB.
249. Fusion Intrabodies as Anti-Huntington Gene Therapy Julie McLear,1,2 Jack Webster,1 Nikolas Webster,1 Anne Messer.1,2 1 Genetic Disorders, Wadsworth Center New York State Dept. of Health, Albany, NY; 2Department of Biomedical Sciences, University at Albany, Albany, NY. Huntington’s Disease (HD) is an autosomal-dominant single gene mutation resulting in a polyglutamine repeat expansion. Pathology results from 37 or more copies of the CAG trinucleotide repeat, with age of onset being inversely related to the number of repeats. Symptoms, usually beginning at mid-life, include motor dysfunction, Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
cognitive and emotional disturbances. Misfolding of the huntingtin protein and formation of high molecular weight molecules have been correlated with brain pathology in humans and mouse models of the disease. Intracellularly expressed single-chain Fv (scFv) antibodies (intrabodies) were engineered to bind adjacent to the expanded polyglutamine, in order to inhibit the pathogenic effects of misfolded mutant huntingtin protein. Intrabodies are small, have a high affinity to antigen and produce a lower immune response compared to traditional extracellular antibodies. Their characteristic increased specificity and potential for genetic engineering make them an ideal candidate for gene therapy. Engineered intrabodies specific for huntingtin have been characterized and show potential in vitro and in vivo, but rescue is incomplete. In order to increase efficacy in vivo, novel fusion intrabodies have now been engineered. Wildtype huntingtin is predominantly localized to the cytoplasm. However, the mutant form, particularly N-terminal fragments, is often nuclear. A nuclear export signal (NES) was recently discovered on the carboxyl-terminus of huntingtin, suggesting that huntingtin can shuttle between the nucleus and cytoplasm. It has been suggested that the NES is lost in mutant huntingtin. Therefore, in order to maintain huntingtin in the cytoplasm, we have introduced intrabody with either an N or C terminal NES. Preliminarily, N terminal NESintrabody showed reduction of aggregates in 3 cell lines, making this a promising intervention. Previous studies suggest that overexpression of the molecular chaperone, heat shock protein 70 (HSP 70), reduces poly Q aggregation in vitro. HSP 70 was therefore fused to intrabody to determine if aggregation was reduced when compared to intrabody expression alone. In cultures, intrabody-HSP 70 fusion protein reduced aggregates more effectively than intrabody or HSP 70 alone, or dual transfections of the two plasmids as separate constructs. Once efficacy of the novel fusion proteins has been further established in vitro, the most promising of these fusion intrabodies will be packaged into viral vectors. The infection and transformation of neurons will be analyzed for reduction of huntingtin pathology. This approach of using fusion intrabodies to counteract the effects of misfolded proteins that are the first step in neurodegeneration is applicable to a series of disorders, including Alzheimer’s, Parkinson’s, and prion diseases. This work is supported by the High Q Foundation and NIH.
250. Protection Against Huntington’s Diease Progression: AAV-Mediated Delivery of Biotherapeutics Adrian P. Kells,1 Rebecca A. Henry,1 Stephanie M. Hughes,1 Richard L. M. Faull,2 Bronwen Connor.1 1 Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand; 2Department of Anatomy, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand. Advances in the development of adeno-associated viral (AAV) vectors for in vivo gene therapy have shown AAV vectors can safely direct the continuous, targeted production of biotherapeutics. This is promising for the treatment of neurological disorders like Huntington’s disease where there is a delayed but progressive degeneration of the caudate-putamen. We have therefore investigated whether AAV-mediated in vivo gene therapy can effectively be used to deliver biotherapeutics and support vulnerable striatal neurons in a rodent model of Huntington’s disease. Our focus is on brainderived neurotrophic factor (BDNF) as a potential biotherapeutic, a protein important for general maintenance and functional plasticity of the striatum. We constructed a recombinant AAV vector with cDNA encoding human BDNF under the control of the chicken beta S95
248. Revealing the Role(s) of TNF-alpha in Early Alzheimer’s Disease Via rAAV-Mediated Hippocampal Gene Transfer in a Triple Transgenic Mouse Model Michelle C. Janelsins,2,4 Michael A. Mastrangelo,4 Keigan M. Park,3,4 Kelly L. Fowlkes,4 Maria Kinsky,4 Wade C. Narrow,4 Salvatore Oddo,5 Frank M. LaFerla,5 Howard J. Federoff,1,2,4 William J. Bowers.1,4 1 Neurology, Univ. Rochester School of Medicine and Dentistry, Rochester, NY; 2Microbiology and Immunology, Univ. Rochester School of Medicine and Dentistry, Rochester, NY; 3Pharmacology and Physiology, Univ. Rochester School of Medicine and Dentistry, Rochester, NY; 4Ctr. for Aging and Devel. Biol., Univ. Rochester School of Medicine and Dentistry, Rochester, NY; 5Neurobiology and Behavior, Univ. California-Irvine, Irvine, CA. Alzheimer’s disease is a complex neurodegenerative disorder that drastically impairs intellectual and emotional functioning in afflicted individuals. The disease is characterized pathologically by a temporal and spatial progression of amyloid-beta (Aβ) deposition, neurofibrillary tangle formation, and synaptic degeneration. Inflammatory mediators have long been proposed as being integral for initiating and/or propagating AD-associated pathologic processes within the brain. To this end, we assessed the expression of proinflammatory molecules in the recently described triple transgenic mouse model (3xTg-AD), which exhibits Aβ deposition, neurofibrillary tangle formation, and synaptic dysfunction with regional specificity similar to human AD. We observed significant up-regulation of the pro-inflammatory cytokine, TNF-α, specifically within one of the earliest affected brain regions in AD, the entorhinal cortex, prior to the onset of overt amyloid pathology. Moreover, the 3xTg-AD entorhinal cortex, unlike the hippocampus, harbored an increased number of microglia/macrophages as determined by quantification of F4/80-positive cells, further implicating the entorhinal cortex in inflammatory processes at early stages of the disease. A recombinant adeno-associated virus (rAAV) vector expressing TNF-α to create a sustained focal inflammatory response was stereotactically delivered to 3xTg-AD mice prior to the appearance of pathological signatures. In ongoing studies, we have observed significant alterations in downstream TNF-α related signaling molecule expression in transduced brain tissues and concomitant modulation of memory/learning behaviors. This integrated biochemical, immunohistochemical, and behavioral approach will provide major insights into the involvement of TNFα mediated inflammation in the temporal and spatial progression of early AD pathogenic events and carries the potential to reveal new therapeutic targets. Supported by NIH F31NS049995 to MCJ, NIH R01AG020204 to HJF, and NIH R01AG023593 to WJB.
249. Fusion Intrabodies as Anti-Huntington Gene Therapy Julie McLear,1,2 Jack Webster,1 Nikolas Webster,1 Anne Messer.1,2 1 Genetic Disorders, Wadsworth Center New York State Dept. of Health, Albany, NY; 2Department of Biomedical Sciences, University at Albany, Albany, NY. Huntington’s Disease (HD) is an autosomal-dominant single gene mutation resulting in a polyglutamine repeat expansion. Pathology results from 37 or more copies of the CAG trinucleotide repeat, with age of onset being inversely related to the number of repeats. Symptoms, usually beginning at mid-life, include motor dysfunction, Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
cognitive and emotional disturbances. Misfolding of the huntingtin protein and formation of high molecular weight molecules have been correlated with brain pathology in humans and mouse models of the disease. Intracellularly expressed single-chain Fv (scFv) antibodies (intrabodies) were engineered to bind adjacent to the expanded polyglutamine, in order to inhibit the pathogenic effects of misfolded mutant huntingtin protein. Intrabodies are small, have a high affinity to antigen and produce a lower immune response compared to traditional extracellular antibodies. Their characteristic increased specificity and potential for genetic engineering make them an ideal candidate for gene therapy. Engineered intrabodies specific for huntingtin have been characterized and show potential in vitro and in vivo, but rescue is incomplete. In order to increase efficacy in vivo, novel fusion intrabodies have now been engineered. Wildtype huntingtin is predominantly localized to the cytoplasm. However, the mutant form, particularly N-terminal fragments, is often nuclear. A nuclear export signal (NES) was recently discovered on the carboxyl-terminus of huntingtin, suggesting that huntingtin can shuttle between the nucleus and cytoplasm. It has been suggested that the NES is lost in mutant huntingtin. Therefore, in order to maintain huntingtin in the cytoplasm, we have introduced intrabody with either an N or C terminal NES. Preliminarily, N terminal NESintrabody showed reduction of aggregates in 3 cell lines, making this a promising intervention. Previous studies suggest that overexpression of the molecular chaperone, heat shock protein 70 (HSP 70), reduces poly Q aggregation in vitro. HSP 70 was therefore fused to intrabody to determine if aggregation was reduced when compared to intrabody expression alone. In cultures, intrabody-HSP 70 fusion protein reduced aggregates more effectively than intrabody or HSP 70 alone, or dual transfections of the two plasmids as separate constructs. Once efficacy of the novel fusion proteins has been further established in vitro, the most promising of these fusion intrabodies will be packaged into viral vectors. The infection and transformation of neurons will be analyzed for reduction of huntingtin pathology. This approach of using fusion intrabodies to counteract the effects of misfolded proteins that are the first step in neurodegeneration is applicable to a series of disorders, including Alzheimer’s, Parkinson’s, and prion diseases. This work is supported by the High Q Foundation and NIH.
250. Protection Against Huntington’s Diease Progression: AAV-Mediated Delivery of Biotherapeutics Adrian P. Kells,1 Rebecca A. Henry,1 Stephanie M. Hughes,1 Richard L. M. Faull,2 Bronwen Connor.1 1 Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand; 2Department of Anatomy, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand. Advances in the development of adeno-associated viral (AAV) vectors for in vivo gene therapy have shown AAV vectors can safely direct the continuous, targeted production of biotherapeutics. This is promising for the treatment of neurological disorders like Huntington’s disease where there is a delayed but progressive degeneration of the caudate-putamen. We have therefore investigated whether AAV-mediated in vivo gene therapy can effectively be used to deliver biotherapeutics and support vulnerable striatal neurons in a rodent model of Huntington’s disease. Our focus is on brainderived neurotrophic factor (BDNF) as a potential biotherapeutic, a protein important for general maintenance and functional plasticity of the striatum. We constructed a recombinant AAV vector with cDNA encoding human BDNF under the control of the chicken beta S95