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280. A Partial Deficiency Model of MUT Methylmalonic Acidemia (MMA) Displays Diet Inducible Disease and Sensitivity to Acetaminophen (APAP)
GENE & CELL THERAPY OF DIABETES, METABOLIC AND GENETIC DISEASES I large scale IS analysis performed on ex vivo transduced cells prior to reinfusion and on engrafted cells by linear amplification mediated PCR (LAM-PCR) and 454 pyrosequencing revealed a polyclonal hematopoietic reconstitution. More than 21.000 unique exactly mappable IS could be retrieved from distinct time points and sorted cell fractions (P1: 6-62 months (M); P2: M6 - M59; P3: M2 - M42; P4: M1 - M16), indicating a highly diverse clonal repertoire. Downstream bioinformatics analysis revealed the characteristic insertion profile reported for lentiviral vectors, showing gene coding regions as preferred targets for lentiviral vector integration (P1: 73.1%; P2: 73.0%; P3: 71.5%; P4: 71.4%). Moreover, a favored integration on chromosomes harboring gene dense regions and regions described for lentiviral clustering, such as KDM2A, PACS1 and HLA genes, has been observed. The landscape of ALD vector integration was very similar in all four patients analyzed. A successful ex vivo transduction of early hematopoietic progenitors is indicated by the detection of identical IS identified in myeloid and lymphoid lineages as well as in CD34+ cells in P1, P2 and P3 (analysis is ongoing for P4). Lentiviral gene therapy shows to be safe and effective, the cerebral disease has been stabilized in 3 out of 4 patients.
280. A Partial Deficiency Model of MUT Methylmalonic Acidemia (MMA) Displays Diet Inducible Disease and Sensitivity to Acetaminophen (APAP)
Julien S. Senac,1 Vijay H. Aswani,2 Justin R. Sysol,1 Irini Manoli,1 Charles P. Venditti.1 1 National Human Genome Research Institute/NIH, Bethesda, MD; 2 Marshfield Clinic, Marshfield, WI. Methylmalonic acidemia (MMA) is caused by mutations of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). We have previously studied MMA using a mouse model mimicking the most severe form of the disease (mut0) that is characterized by a complete absence of Mut. Mut-/- mice are fragile, display immediate lethality and hence, do not live long enough to study disease related manifestations, such as metabolic stroke, pancreatitis, kidney disease and pharmacogenomic suspectibilities. To overcome these difficulties, we developed transgenic mice that express the Mut enzyme in a tissuespecific fashion, however, these animals restrict the study of MMA to selected organs. Among the wide spectrum of partial activity or mutmutations, the mechanism of enzymatic impairment has been defined only for a few, such as p.G717V, a mutation that has been documented to be an adenosylcobalamin Km mutant. Following physiologic and phenotypic characterization of transgenic mice that ubiquitously express the mutant homologue (p.G715V) as a stable transgene (Mut/;TgINS-CBA-G715V), we demonstrate that this new murine model mimics the physiologic and phenotypic manifestations observed in mut- MMA patients, such as the inducible formation of megamitochondria in the liver. These mice have enabled a new inducible platform to study the pharmacogenetic effects of common medicines used by MMA patients that are potentially hepatotoxic, such as acetaminophen (APAP). Following studies previously published on APAP in mice, Mut-/-;TgINS-CBA-G715V mice (n=8) and heterozygous littermates (n=6) were fasted overnight and then injected via the intraperitoneal route with a single dose of 300 mg/kg of APAP. Mice were monitored over a period of 24 hours and blood samples collected 3, 6, 12, and 24 hours post-injection. Heterozygous mice displayed normal to moderately elevated ALT measurements, while Mut-/-;TgINS-CBA-G715V mice (n=8) exhibited much larger elevations of ALT (6-fold increase vs het; p-value = 0.001) with associated mortality as 1 out of 6 mice perished after the APAP injection. In order to examine the effect of Mut expression in the resistance to APAP treatment, we injected Mut-/-;TgINS-CBA-G715V mice with rAAV8-TBG-Mut at a dose of 1e10 GC per gram and repeated the APAP experiment with a similar dose of 300 mg/kg two weeks after AAV administration. After gene therapy, Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
the Mut-/-;TgINS-CBA-G715V mice (n=8) showed lower elevations of ALT after APAP challenge compared to the untreated state (4 fold lower vs untreated; p=0.001) and increased survival, suggesting that Mut deficiency confers a pharmocogenomic susceptibility to APAP that appears to be mediated by a cell-intrinsic mechanism. APAP exposure might provide a facile means to increase hepatic engraftment of wildtype or gene-corrected cells in the setting of MMA, and possibly other disorders that exhibit secondary mitochondrial dysfunction in the hepatocyte.
281. AAV-Based over Expression of PEPCK-C Re-Patterns Mouse Muscle Energy Metabolism and Prevents High-Fat Diet Induced Obesity
Daibang Nie,1 Sheri Wang,1 Ying Tang,1 Dong Wei,2 William Witt,3 Allan Zhao,2 Yifan Dai,3 Freddie Fu,1 Johnny Huard,1 Bing Wang.1 1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 2 Cell Biology & Physiology, University of Pittsburgh, Pittsburgh, PA; 3Transplantation Institute, University of Pittsburgh, Pittsburgh, PA. Phosphoenolpyruvate Carboxykinase, cytosolic form (PEPCK-C) is a key enzyme involving energy metabolism. However, the low level of PEPCK-C expression in mammalian skeletal muscle leads to the use of glycogen as the dominant source of energy (2xATP) during intensive exercise. Over-expression of PEPCK-C in skeletal muscle of transgenic mice re-patterns energy metabolism by using fatty acids as the primary source of energy (6xATP) for physical activity, decreasing the level of fatty acids in the blood circulation and fat mass. However, a transgenic model cannot always be applied to clinical events. Here, we tested the hypothesis that recombinant adeno-associated viral (rAAV) vector-mediated PEPCK-C over expression in muscle alters energy metabolism, decreasing fatty acids circulating in the blood. An rAAV8 vector carrying the PEPCK-C or GFP gene under the control of a muscle specific promoter was purified as standard protocol. A single intraperitoneal (i.p.) injection (3x1011 v.g. particles) of AAV8-tMCKGFP or AAV8-tMCK-PEPCK-C was given to 10-day old C57BL/6J mice, 16 neonates for each i.p. treatment group. The high-fat diet for generating obese mice started at 1 month post-rAAV treatment and continuously fed for 8 weeks. PBS treatment served as control. The level of PEPCK-C in muscle was determined 1 month post-treatment by western blot analysis. Glucose and insulin tolerance tests were tested at 10 weeks of age. Muscle mitochondria and GFP expression were analyzed at 5 months of age. In induced obesity (high-fat diet) experiment, mitochondria were also analyzed. Treadmill tests were performed at 6 and 9 weeks of age to evaluate muscle endurance. We found that both GFP reporter and PEPCK-C were expressed in skeletal muscle of treated mice one month post-treatment, but not in liver. Expression of PEPCK-C in muscle did not affect glucose or insulin tolerance of animals at 10 weeks of age. An increase in muscle mitochondrial density and size was found in PCPCK-C treated thigh muscle in EM (Mag. 25K) at 5 months of age. The endurance on treadmill showed a 1.5-fold increase in PEPCK-C treated mice at both 6 and 9 weeks of age (* p <0.005). Improved mitochondria and reduced fat mass were found in the PEPCK-C treated mice fed by high-fat diet. The PBS control showed the high-fat diet induced fat mass and damaged mitochondria, as observed in the muscle of obese patents. This study demonstrates that skeletal muscle could be targeted as the organ system to over-express PEPCK-C, which enhances muscle endurance and prevents obesity by altering energy metabolism and using fatty acids as a source in mitochondria. In further study, we will test whether the specific over-expression of PEPCK-C has potentially therapeutic functions for the prevention and treatment of type 2 diabetes mellitus.
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280. A Partial Deficiency Model of MUT Methylmalonic Acidemia (MMA) Displays Diet Inducible Disease and Sensitivity to Acetaminophen (APAP)
Julien S. Senac,1 Vijay H. Aswani,2 Justin R. Sysol,1 Irini Manoli,1 Charles P. Venditti.1 1 National Human Genome Research Institute/NIH, Bethesda, MD; 2 Marshfield Clinic, Marshfield, WI. Methylmalonic acidemia (MMA) is caused by mutations of the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). We have previously studied MMA using a mouse model mimicking the most severe form of the disease (mut0) that is characterized by a complete absence of Mut. Mut-/- mice are fragile, display immediate lethality and hence, do not live long enough to study disease related manifestations, such as metabolic stroke, pancreatitis, kidney disease and pharmacogenomic suspectibilities. To overcome these difficulties, we developed transgenic mice that express the Mut enzyme in a tissuespecific fashion, however, these animals restrict the study of MMA to selected organs. Among the wide spectrum of partial activity or mutmutations, the mechanism of enzymatic impairment has been defined only for a few, such as p.G717V, a mutation that has been documented to be an adenosylcobalamin Km mutant. Following physiologic and phenotypic characterization of transgenic mice that ubiquitously express the mutant homologue (p.G715V) as a stable transgene (Mut/;TgINS-CBA-G715V), we demonstrate that this new murine model mimics the physiologic and phenotypic manifestations observed in mut- MMA patients, such as the inducible formation of megamitochondria in the liver. These mice have enabled a new inducible platform to study the pharmacogenetic effects of common medicines used by MMA patients that are potentially hepatotoxic, such as acetaminophen (APAP). Following studies previously published on APAP in mice, Mut-/-;TgINS-CBA-G715V mice (n=8) and heterozygous littermates (n=6) were fasted overnight and then injected via the intraperitoneal route with a single dose of 300 mg/kg of APAP. Mice were monitored over a period of 24 hours and blood samples collected 3, 6, 12, and 24 hours post-injection. Heterozygous mice displayed normal to moderately elevated ALT measurements, while Mut-/-;TgINS-CBA-G715V mice (n=8) exhibited much larger elevations of ALT (6-fold increase vs het; p-value = 0.001) with associated mortality as 1 out of 6 mice perished after the APAP injection. In order to examine the effect of Mut expression in the resistance to APAP treatment, we injected Mut-/-;TgINS-CBA-G715V mice with rAAV8-TBG-Mut at a dose of 1e10 GC per gram and repeated the APAP experiment with a similar dose of 300 mg/kg two weeks after AAV administration. After gene therapy, Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy
the Mut-/-;TgINS-CBA-G715V mice (n=8) showed lower elevations of ALT after APAP challenge compared to the untreated state (4 fold lower vs untreated; p=0.001) and increased survival, suggesting that Mut deficiency confers a pharmocogenomic susceptibility to APAP that appears to be mediated by a cell-intrinsic mechanism. APAP exposure might provide a facile means to increase hepatic engraftment of wildtype or gene-corrected cells in the setting of MMA, and possibly other disorders that exhibit secondary mitochondrial dysfunction in the hepatocyte.
281. AAV-Based over Expression of PEPCK-C Re-Patterns Mouse Muscle Energy Metabolism and Prevents High-Fat Diet Induced Obesity
Daibang Nie,1 Sheri Wang,1 Ying Tang,1 Dong Wei,2 William Witt,3 Allan Zhao,2 Yifan Dai,3 Freddie Fu,1 Johnny Huard,1 Bing Wang.1 1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 2 Cell Biology & Physiology, University of Pittsburgh, Pittsburgh, PA; 3Transplantation Institute, University of Pittsburgh, Pittsburgh, PA. Phosphoenolpyruvate Carboxykinase, cytosolic form (PEPCK-C) is a key enzyme involving energy metabolism. However, the low level of PEPCK-C expression in mammalian skeletal muscle leads to the use of glycogen as the dominant source of energy (2xATP) during intensive exercise. Over-expression of PEPCK-C in skeletal muscle of transgenic mice re-patterns energy metabolism by using fatty acids as the primary source of energy (6xATP) for physical activity, decreasing the level of fatty acids in the blood circulation and fat mass. However, a transgenic model cannot always be applied to clinical events. Here, we tested the hypothesis that recombinant adeno-associated viral (rAAV) vector-mediated PEPCK-C over expression in muscle alters energy metabolism, decreasing fatty acids circulating in the blood. An rAAV8 vector carrying the PEPCK-C or GFP gene under the control of a muscle specific promoter was purified as standard protocol. A single intraperitoneal (i.p.) injection (3x1011 v.g. particles) of AAV8-tMCKGFP or AAV8-tMCK-PEPCK-C was given to 10-day old C57BL/6J mice, 16 neonates for each i.p. treatment group. The high-fat diet for generating obese mice started at 1 month post-rAAV treatment and continuously fed for 8 weeks. PBS treatment served as control. The level of PEPCK-C in muscle was determined 1 month post-treatment by western blot analysis. Glucose and insulin tolerance tests were tested at 10 weeks of age. Muscle mitochondria and GFP expression were analyzed at 5 months of age. In induced obesity (high-fat diet) experiment, mitochondria were also analyzed. Treadmill tests were performed at 6 and 9 weeks of age to evaluate muscle endurance. We found that both GFP reporter and PEPCK-C were expressed in skeletal muscle of treated mice one month post-treatment, but not in liver. Expression of PEPCK-C in muscle did not affect glucose or insulin tolerance of animals at 10 weeks of age. An increase in muscle mitochondrial density and size was found in PCPCK-C treated thigh muscle in EM (Mag. 25K) at 5 months of age. The endurance on treadmill showed a 1.5-fold increase in PEPCK-C treated mice at both 6 and 9 weeks of age (* p <0.005). Improved mitochondria and reduced fat mass were found in the PEPCK-C treated mice fed by high-fat diet. The PBS control showed the high-fat diet induced fat mass and damaged mitochondria, as observed in the muscle of obese patents. This study demonstrates that skeletal muscle could be targeted as the organ system to over-express PEPCK-C, which enhances muscle endurance and prevents obesity by altering energy metabolism and using fatty acids as a source in mitochondria. In further study, we will test whether the specific over-expression of PEPCK-C has potentially therapeutic functions for the prevention and treatment of type 2 diabetes mellitus.
S107