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821. Neprilysin Gene Transfer Reduces Amyloid Pathology in Transgenic Mouse Models of Alzheimer's Disease
GENE THERAPY FOR THE NERVOUS SYSTEM I GENE THERAPY FOR THE NERVOUS SYSTEM I 821. Neprilysin Gene Transfer Reduces Amyloid Pathology in Transgenic Mouse Models of Alzheimer’s Disease Robert A. Marr,1 Edward Rockenstien,2 Atish Mukherjee,3 Mark S. Kindy,3 Louis B. Hersh,3 Fred H. Gage,1 Inder M. Verma,1 Eliezer Masliah.2 1 Laboratory of Genetics, The Salk Institute, La Jolla, CA, United States; 2Department of Neurosciences, The University of California San Diego, La Jolla, CA, United States; 3Department of Biochemistry, The University of Kentucky, Lexington, KY, United States. The degenerative process of Alzheimer’s disease is linked to the accumulation of amyloid-β (Aβ) peptides in the brain. Neprilysin has recently been implicated as a major extracellular Aβ degrading enzyme which mediates catabolism of Aβ in mice. We set out to test if gene delivery of neprilysin to the effected areas of the brain could be therapeutic in mouse models of amyloidosis. A lentiviral vector expressing human neprilysin (Lenti-Nep) was constructed and shown to efficiently express biologically active protein. We show that unilateral intra-cerebral injection of Lenti-Nep reduced amyloidβ deposits by half relative to the untreated side. Furthermore, LentiNep ameliorated neurodegenerative alterations in the frontal cortex and hippocampus of these transgenic mice as shown by MAP2 immunoreactivity. These data further support a role for neprilysin in regulating cerebral amyloid deposition and demonstrates a new potential gene-therapeutic approach to Alzheimer’s disease therapy.
822.
Silencing Huntingtin with siRNA
Scott Q. Harper,1 Patrick D. Staber,1 Sarah K. Fineberg,1 Henry L. Paulson,1,2 Beverly L. Davidson.1,3 1 Internal Medicine; 2Neurology; 3Physiology & Biophysics, University of Iowa, Iowa City, IA. Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by polyglutamine expansion in huntingtin protein. This gain of function mutation results in striatal and cortical neuron degeneration leading to severe neurological impairment and death. An effective treatment will require a reduction of mutant huntingtin protein. Recent advances by this laboratory and others have demonstrated the feasibility of treating dominant genetic disorders using siRNA (small inhibitory RNA). Using in vitro synthesized siRNA and plasmids expressing hairpin siRNAs from the U6 promoter, we tested 10 different siRNAs corresponding to various regions of the human huntingtin transcript (siHDs). U6 plasmids or siRNAs were cotransfected with target plasmids expressing either the full-length normal human huntingtin cDNA or a truncated transcript encoding exons 1-3 and containing an expanded polyglutamine tract (hHunt-82Q). RNA and protein were harvested from transfected cells 48 hrs later, and siRNA mediated silencing was assayed by phosphorimager quantification of northern and western blots. We observed a dose-dependent reduction in huntingtin expression with some non-specific effects at the highest ratios of siRNA:target tested. To date, our most effective U6driven siHD reduce hHunt-82Q mRNA and protein levels in cell culture by 86% and 55% respectively. We are currently developing viral vectors to deliver our most effective siHDs to brains of HD mouse models.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
823. Characterizing Pol II and Pol III Driven siRNA: Application to Spinal Cerebellar Ataxia Type I Haibin Xia,1 Qinwen Mao,1 Nathan Kiewiet,1 Beverly L. Davidson.1,3 1 Internal Medicine; 2Neurology; 3Physiology & Biophysics, University of Iowa, Iowa City, IA. siRNA has been widely used for gene silencing in vitro, and more recently in vivo. In most instances, siRNA expression has been driven from ubiquitously expressed Pol III promoters such as the U6 or H1 promoters. In prior work, we found that a modified CMV (Pol II) promoter, combined with a minimal polyA cassette, could successfully drive siRNA expression for efficient diminution in target sequence expression. In this study, we compared the levels of siRNA expressed from the two promoters, and the cellular location of the expressed siRNA. We also tested the effects of hairpin length, distance between promoter and hairpin, and between hairpin and polyA, on siRNA expression levels and gene silencing. In Pol II and Pol III promoter comparison studies, we found that target diminution was modestly increased when the siRNA was expressed from a Pol III promoter, even though the levels of total RNA expressed were much higher. Fractionation studies revealed that Pol II expressed siRNA was found predominately in the cytoplasm, while most Pol III expressed siRNA was in the nucleus. In studies on the Pol II promoter system, we identified 6 bp as the maximal amount of flanking sequence tolerated 5’ and 3’ of the hairpin, and found that 19 or 21 bp hairpins were optimal for gene silencing. These characteristics were used to build siRNA against ataxin-1. Spinal cerebellar ataxia type I is a classical CAG repeat disease, due to a polyglutamine expansion in ataxin-1; therapies would require silencing gene expression. Ataxin-1 specific siRNAs (F1 through F11) were generated. One F11 led to very effective gene silencing as measured by western blot. Our data support the utility of Pol II promoters for gene silencing studies, with application to an important class of dominant inherited disorders.
824. AAV-Mediated Gene Therapy for Murine Infantile Neuronal Ceroid Lipofuscinosis (INCL, Batten Disease) M. Griffey,1 E. Bible,2 J. Cooper,2 D. Wozniak,3 S. Rothman,4 C. Vogler,5 M. Sands.1 1 Internal Medicine, Washington University Medical School, St. Louis, MO, United States; 2Neuropathology, King’s College, London, United Kingdom; 3Psychiatry, Washington University Medical School, St. Louis, MO, United States; 4Neurology, Washington University School of Medicine, St. Louis, MO, United States; 5Pathology, St. Louis University, St. Louis, MO, United States. The Neruonal Ceroid Liposfuscinoses (NCL, Batten Disease) are a group of inherited pediatric neurodegenerative diseases. Children with NCL suffer from blindness, seizures, a decline in motor and mental abilities, and finally premature death. Autofluorescent storage material accumulates in many cell types, including neurons. This accumulation leads to neuronal loss and widespread brain atrophy. The earliest form of NCL has an onset at 1.5 years of age and is known as Infantile Neuronal Ceroid Lipofuscinosis (INCL). The primary defect in INCL is due to mutations in the gene that encodes the lysosomal hydrolase palmitoyl protein thioesterase 1 (PPT1). A murine model of PPT1-deficiency that exhibits many of the same clinical signs as humans with INCL was recently created by Dr. S. Hofmann’s group (University of Texas Southwestern). To determine the efficacy of a viral-mediated gene therapy approach to treat the PPT1-deficient mouse we created a recombinant adeno-associated viral (AAV) vector containing the human PPT1 cDNA and tested its S317
822.
Silencing Huntingtin with siRNA
Scott Q. Harper,1 Patrick D. Staber,1 Sarah K. Fineberg,1 Henry L. Paulson,1,2 Beverly L. Davidson.1,3 1 Internal Medicine; 2Neurology; 3Physiology & Biophysics, University of Iowa, Iowa City, IA. Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by polyglutamine expansion in huntingtin protein. This gain of function mutation results in striatal and cortical neuron degeneration leading to severe neurological impairment and death. An effective treatment will require a reduction of mutant huntingtin protein. Recent advances by this laboratory and others have demonstrated the feasibility of treating dominant genetic disorders using siRNA (small inhibitory RNA). Using in vitro synthesized siRNA and plasmids expressing hairpin siRNAs from the U6 promoter, we tested 10 different siRNAs corresponding to various regions of the human huntingtin transcript (siHDs). U6 plasmids or siRNAs were cotransfected with target plasmids expressing either the full-length normal human huntingtin cDNA or a truncated transcript encoding exons 1-3 and containing an expanded polyglutamine tract (hHunt-82Q). RNA and protein were harvested from transfected cells 48 hrs later, and siRNA mediated silencing was assayed by phosphorimager quantification of northern and western blots. We observed a dose-dependent reduction in huntingtin expression with some non-specific effects at the highest ratios of siRNA:target tested. To date, our most effective U6driven siHD reduce hHunt-82Q mRNA and protein levels in cell culture by 86% and 55% respectively. We are currently developing viral vectors to deliver our most effective siHDs to brains of HD mouse models.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
823. Characterizing Pol II and Pol III Driven siRNA: Application to Spinal Cerebellar Ataxia Type I Haibin Xia,1 Qinwen Mao,1 Nathan Kiewiet,1 Beverly L. Davidson.1,3 1 Internal Medicine; 2Neurology; 3Physiology & Biophysics, University of Iowa, Iowa City, IA. siRNA has been widely used for gene silencing in vitro, and more recently in vivo. In most instances, siRNA expression has been driven from ubiquitously expressed Pol III promoters such as the U6 or H1 promoters. In prior work, we found that a modified CMV (Pol II) promoter, combined with a minimal polyA cassette, could successfully drive siRNA expression for efficient diminution in target sequence expression. In this study, we compared the levels of siRNA expressed from the two promoters, and the cellular location of the expressed siRNA. We also tested the effects of hairpin length, distance between promoter and hairpin, and between hairpin and polyA, on siRNA expression levels and gene silencing. In Pol II and Pol III promoter comparison studies, we found that target diminution was modestly increased when the siRNA was expressed from a Pol III promoter, even though the levels of total RNA expressed were much higher. Fractionation studies revealed that Pol II expressed siRNA was found predominately in the cytoplasm, while most Pol III expressed siRNA was in the nucleus. In studies on the Pol II promoter system, we identified 6 bp as the maximal amount of flanking sequence tolerated 5’ and 3’ of the hairpin, and found that 19 or 21 bp hairpins were optimal for gene silencing. These characteristics were used to build siRNA against ataxin-1. Spinal cerebellar ataxia type I is a classical CAG repeat disease, due to a polyglutamine expansion in ataxin-1; therapies would require silencing gene expression. Ataxin-1 specific siRNAs (F1 through F11) were generated. One F11 led to very effective gene silencing as measured by western blot. Our data support the utility of Pol II promoters for gene silencing studies, with application to an important class of dominant inherited disorders.
824. AAV-Mediated Gene Therapy for Murine Infantile Neuronal Ceroid Lipofuscinosis (INCL, Batten Disease) M. Griffey,1 E. Bible,2 J. Cooper,2 D. Wozniak,3 S. Rothman,4 C. Vogler,5 M. Sands.1 1 Internal Medicine, Washington University Medical School, St. Louis, MO, United States; 2Neuropathology, King’s College, London, United Kingdom; 3Psychiatry, Washington University Medical School, St. Louis, MO, United States; 4Neurology, Washington University School of Medicine, St. Louis, MO, United States; 5Pathology, St. Louis University, St. Louis, MO, United States. The Neruonal Ceroid Liposfuscinoses (NCL, Batten Disease) are a group of inherited pediatric neurodegenerative diseases. Children with NCL suffer from blindness, seizures, a decline in motor and mental abilities, and finally premature death. Autofluorescent storage material accumulates in many cell types, including neurons. This accumulation leads to neuronal loss and widespread brain atrophy. The earliest form of NCL has an onset at 1.5 years of age and is known as Infantile Neuronal Ceroid Lipofuscinosis (INCL). The primary defect in INCL is due to mutations in the gene that encodes the lysosomal hydrolase palmitoyl protein thioesterase 1 (PPT1). A murine model of PPT1-deficiency that exhibits many of the same clinical signs as humans with INCL was recently created by Dr. S. Hofmann’s group (University of Texas Southwestern). To determine the efficacy of a viral-mediated gene therapy approach to treat the PPT1-deficient mouse we created a recombinant adeno-associated viral (AAV) vector containing the human PPT1 cDNA and tested its S317