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459. Gene Transfer of Constitutively Active Akt1 Increases Islet Cell Survival and Proliferation
GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II 458. Gene Therapy with Small Heat Shock Protein for Huntington Disease
Wuh-Liang Hwu,1 Yu-May Lee,2 Nathalie Clement,3 Ni-Chung Lee,1 Barry Byrne.3 1 Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan; 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 3Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL. Huntington disease (HD) is a late-onset neurodegenerative disease characterized by intranuclear and cytoplasmic aggregates and cell death in the striatal region. HD is caused by abnormal expansion of the trinucleotide (CAG) repeat sequence in exon 1 of the gene (HTT) encoding the huntingtin protein. Recently, heat shock proteins are identified as potential modulators of poly(Q) aggregation and/or cell death. More interestingly, the small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) protect cells from heat shock and oxidative stress, and decrease apoptosis. Cells with expanded poly(Q) revealed reduced protein expression of HSP27. In this study, we use AAV1 vector to design a gene therapy for Huntington’s disease. Intracerebral injection of AAV1 virus has been shown to give wide spread expression in mouse brain. We first injected 2 x 109 vg of scAAV1-CMV- mCherry to the putamen of control mice. Two week after injection, we could demonstrate widespread expression of red fluorescence on the injection hemisphere but not in the controlateral side. We then packaged AAV1-CMV-Hsp27 (1 x 1012 vg/ml). HD R6/2 mice were then injected with AAV1-CMV-Hsp27 (2 x 109 vg per site) or saline at bilateral putamens at 6 weeks of age. These animals were followed by rotorod analysis weekly. After 4 weeks post-injection, the treated mice showed poorer performance on rotorod than the mock-injected mice. It is possible that overexpression of the molecular chaperones may disturb normal protein turn over in the cells and then brings to untoward effects. It may be necessary to control the expression level in chaperone therapy in the future.
459. Gene Transfer of Constitutively Active Akt1 Increases Islet Cell Survival and Proliferation
Robert N. Bone,1 Yuan Zhang,2 Mert Icyuz,2 Stacie M. J. Bryant,3 Henry K. Fortinberry,3 John A. Thompson,3 Gene P. Siegal,1 Hongju Wu.4 1 Pathology, University of Alabama at Birmingham, Birmingham, AL; 2Medicine, University of Alabama at Birmingham, Birmingham, AL; 3Surgery, University of Alabama at Birmingham, Birmingham, AL; 4Ostetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL.
Type 1 Diabetes mellitus (T1D) is a metabolic disorder characterized by an absolute insulin deficiency due to the autoimmune destruction of insulin producing beta cells. Exogenous insulin therapy is the main treatment; however, it does not cure T1D. Islet transplantation is becoming a viable option to cure T1D. However, limited islet supply, inefficient islet recovery, and significant islet loss restrict the success of transplantation therapy. Therefore, a means to promote beta cell survival and proliferation is expected to offer therapeutic benefits. Akt1/PKB, a serine/theronine kinase that is the direct downstream target of PI3 kinase, has been shown to have anti-apoptotic function as well as inducing cell proliferation. Relevance of Akt1 to diabetes has been shown in transgenic mice expressing constitutively active Akt1 (CA-Akt1) and knockout mice lacking one form of Akt. On these basis, we hypothesize that gene transfer of CA-Akt1 into beta cells will promote beta cell survival and proliferation, thus can be protective to experimental diabetes. In this study, we generated a highly efficient and specific vector that can deliver CA-Akt1 into islet beta cells. The vector is based on a fiber-modified Ad5, Ad5RGDpk7, which has been shown to have enhanced infectivity for islet cells. In order to drive specificity to the pancreatic beta cells, the beta cell specific rat insulin promoter was used to control CA-Akt1 expression. Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Our western blotting data have shown that this vector can efficiently deliver CA-Akt1 into islet cells in isolated human, rat and porcine islets. Islets transduced with the CA-Akt1 vector showed enhanced viability compared to control groups. Using BrdU incorporation assay, we found CA-Akt1 expression in the islet cells promoted beta cell proliferation. We also examined if CA-Akt1 expression could protect islet cells in vivo in streptozotocin-induced diabetic mice. Our data have shown the mice expressing CA-Akt1 exhibited better blood glucose control daily and following an single dose glucose challenge. We also found the pancreas of the CA-Akt1 treated mice contained more islets upon sacrifice. Taken together, our data suggest CA-Akt1 is effective in promoting beta cell survival and proliferation, thus has potential therapeutic use.
460. Helper-Dependent Adenoviral Vectors for Liver-Directed Gene Therapy of Primary Hyperoxaluria Type 1
Raffaele Castello,1 Pasquale Piccolo,1 Roberta Borzone,1 Riccardo Sangermano,1 Donna Palmer,2 Philip Ng,2 Nicola Brunetti-Pierri.1,3 1 Telethon Institute of Genetics and Medicine, Naples, Italy; 2 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3Pediatrics, Federico II University, Naples, Italy. Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT), which catalyzes the conversion of glyoxylate to glycine. AGT deficiency results in overproduction of oxalate which forms insoluble calcium salts that accumulate in the kidney and other organs which ultimately lead to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/ kidney transplantation is the only therapeutic strategy available to prevent kidney failure. However, this treatment is associated with significant mortality and morbidity. Gene therapy is an attractive option to provide a definitive cure for PH1. Towards this goal, we are investigating helper-dependent adenoviral (HDAd) vectors for liverdirected gene therapy of PH1. HDAd vectors are very attractive for gene therapy because they are devoid of all viral coding sequences and result in efficient hepatocyte transduction and long-term transgene expression without chronic toxicity. We have injected PH1 mice with an HDAd encoding the AGT under the control of the liver-specific phosphoenolpyruvate carboxykinase (PEPCK) promoter and observed a sustained reduction of oxalate urinary excretion. Recently, we have developed a minimally invasive method to improve the therapeutic index of HDAd (Brunetti-Pierri et al., 2009). This approach based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver, results in substantially higher efficiency of hepatocyte transduction using clinically relevant low vector doses and is accompanied by mild-to-moderate acute but transient toxicities. Therefore, this method of vector delivery may permits correction of PH1 using clinically relevant doses of HDAd and may thus pave the way to clinical application of HDAd for PH1 gene therapy.
461. Administration of Gene Therapy Improves Cholesterol Synthesis and Physical Outcome in a Mouse Model of Smith-Lemli-Opitz Syndrome (SLOS)
Lee Ying,1 Xavier Matabosch,1 Montserrat Serra,1 Cedric Shackleton,1 Gordon Watson.1 1 Children’s Hospital Oakland Research Institute, Oakland, CA.
Smith-Lemli-Opitz syndrome is an inborn error of cholesterol biosynthesis that results in a range of symptoms, including cognitive impairments, physical dysmorphia, and delayed growth. The biochemical cause is a defect in 7-dehydrocholesterol reductase (DHCR7), the enzyme that converts 7-dehydrocholesterol (7DHC) to S177
Wuh-Liang Hwu,1 Yu-May Lee,2 Nathalie Clement,3 Ni-Chung Lee,1 Barry Byrne.3 1 Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan; 2Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; 3Pediatrics, Powell Gene Therapy Center, University of Florida, Gainesville, FL. Huntington disease (HD) is a late-onset neurodegenerative disease characterized by intranuclear and cytoplasmic aggregates and cell death in the striatal region. HD is caused by abnormal expansion of the trinucleotide (CAG) repeat sequence in exon 1 of the gene (HTT) encoding the huntingtin protein. Recently, heat shock proteins are identified as potential modulators of poly(Q) aggregation and/or cell death. More interestingly, the small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) protect cells from heat shock and oxidative stress, and decrease apoptosis. Cells with expanded poly(Q) revealed reduced protein expression of HSP27. In this study, we use AAV1 vector to design a gene therapy for Huntington’s disease. Intracerebral injection of AAV1 virus has been shown to give wide spread expression in mouse brain. We first injected 2 x 109 vg of scAAV1-CMV- mCherry to the putamen of control mice. Two week after injection, we could demonstrate widespread expression of red fluorescence on the injection hemisphere but not in the controlateral side. We then packaged AAV1-CMV-Hsp27 (1 x 1012 vg/ml). HD R6/2 mice were then injected with AAV1-CMV-Hsp27 (2 x 109 vg per site) or saline at bilateral putamens at 6 weeks of age. These animals were followed by rotorod analysis weekly. After 4 weeks post-injection, the treated mice showed poorer performance on rotorod than the mock-injected mice. It is possible that overexpression of the molecular chaperones may disturb normal protein turn over in the cells and then brings to untoward effects. It may be necessary to control the expression level in chaperone therapy in the future.
459. Gene Transfer of Constitutively Active Akt1 Increases Islet Cell Survival and Proliferation
Robert N. Bone,1 Yuan Zhang,2 Mert Icyuz,2 Stacie M. J. Bryant,3 Henry K. Fortinberry,3 John A. Thompson,3 Gene P. Siegal,1 Hongju Wu.4 1 Pathology, University of Alabama at Birmingham, Birmingham, AL; 2Medicine, University of Alabama at Birmingham, Birmingham, AL; 3Surgery, University of Alabama at Birmingham, Birmingham, AL; 4Ostetrics and Gynecology, University of Alabama at Birmingham, Birmingham, AL.
Type 1 Diabetes mellitus (T1D) is a metabolic disorder characterized by an absolute insulin deficiency due to the autoimmune destruction of insulin producing beta cells. Exogenous insulin therapy is the main treatment; however, it does not cure T1D. Islet transplantation is becoming a viable option to cure T1D. However, limited islet supply, inefficient islet recovery, and significant islet loss restrict the success of transplantation therapy. Therefore, a means to promote beta cell survival and proliferation is expected to offer therapeutic benefits. Akt1/PKB, a serine/theronine kinase that is the direct downstream target of PI3 kinase, has been shown to have anti-apoptotic function as well as inducing cell proliferation. Relevance of Akt1 to diabetes has been shown in transgenic mice expressing constitutively active Akt1 (CA-Akt1) and knockout mice lacking one form of Akt. On these basis, we hypothesize that gene transfer of CA-Akt1 into beta cells will promote beta cell survival and proliferation, thus can be protective to experimental diabetes. In this study, we generated a highly efficient and specific vector that can deliver CA-Akt1 into islet beta cells. The vector is based on a fiber-modified Ad5, Ad5RGDpk7, which has been shown to have enhanced infectivity for islet cells. In order to drive specificity to the pancreatic beta cells, the beta cell specific rat insulin promoter was used to control CA-Akt1 expression. Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
Our western blotting data have shown that this vector can efficiently deliver CA-Akt1 into islet cells in isolated human, rat and porcine islets. Islets transduced with the CA-Akt1 vector showed enhanced viability compared to control groups. Using BrdU incorporation assay, we found CA-Akt1 expression in the islet cells promoted beta cell proliferation. We also examined if CA-Akt1 expression could protect islet cells in vivo in streptozotocin-induced diabetic mice. Our data have shown the mice expressing CA-Akt1 exhibited better blood glucose control daily and following an single dose glucose challenge. We also found the pancreas of the CA-Akt1 treated mice contained more islets upon sacrifice. Taken together, our data suggest CA-Akt1 is effective in promoting beta cell survival and proliferation, thus has potential therapeutic use.
460. Helper-Dependent Adenoviral Vectors for Liver-Directed Gene Therapy of Primary Hyperoxaluria Type 1
Raffaele Castello,1 Pasquale Piccolo,1 Roberta Borzone,1 Riccardo Sangermano,1 Donna Palmer,2 Philip Ng,2 Nicola Brunetti-Pierri.1,3 1 Telethon Institute of Genetics and Medicine, Naples, Italy; 2 Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 3Pediatrics, Federico II University, Naples, Italy. Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of the peroxisomal enzyme alanine:glyoxylate-aminotransferase (AGT), which catalyzes the conversion of glyoxylate to glycine. AGT deficiency results in overproduction of oxalate which forms insoluble calcium salts that accumulate in the kidney and other organs which ultimately lead to end-stage renal disease and death. Organ transplantation as either preemptive liver transplantation or combined liver/ kidney transplantation is the only therapeutic strategy available to prevent kidney failure. However, this treatment is associated with significant mortality and morbidity. Gene therapy is an attractive option to provide a definitive cure for PH1. Towards this goal, we are investigating helper-dependent adenoviral (HDAd) vectors for liverdirected gene therapy of PH1. HDAd vectors are very attractive for gene therapy because they are devoid of all viral coding sequences and result in efficient hepatocyte transduction and long-term transgene expression without chronic toxicity. We have injected PH1 mice with an HDAd encoding the AGT under the control of the liver-specific phosphoenolpyruvate carboxykinase (PEPCK) promoter and observed a sustained reduction of oxalate urinary excretion. Recently, we have developed a minimally invasive method to improve the therapeutic index of HDAd (Brunetti-Pierri et al., 2009). This approach based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver, results in substantially higher efficiency of hepatocyte transduction using clinically relevant low vector doses and is accompanied by mild-to-moderate acute but transient toxicities. Therefore, this method of vector delivery may permits correction of PH1 using clinically relevant doses of HDAd and may thus pave the way to clinical application of HDAd for PH1 gene therapy.
461. Administration of Gene Therapy Improves Cholesterol Synthesis and Physical Outcome in a Mouse Model of Smith-Lemli-Opitz Syndrome (SLOS)
Lee Ying,1 Xavier Matabosch,1 Montserrat Serra,1 Cedric Shackleton,1 Gordon Watson.1 1 Children’s Hospital Oakland Research Institute, Oakland, CA.
Smith-Lemli-Opitz syndrome is an inborn error of cholesterol biosynthesis that results in a range of symptoms, including cognitive impairments, physical dysmorphia, and delayed growth. The biochemical cause is a defect in 7-dehydrocholesterol reductase (DHCR7), the enzyme that converts 7-dehydrocholesterol (7DHC) to S177