185. Muscle Specific Gene Transfer of the PEPCK-C Improves Physical Activity in Mice

METABOLIC DISEASES I not integrate efficiently in any organ. The mechanism underlying the successful treatment of cystinosis by BMC or HSC transplantation is the successful engraftment of cells expressing a functional Ctns gene. Indeed, we showed that mice transplanted under otherwise identical conditions with Ctns-/- BMC still accumulate cystine and develop all the tissue injuries including renal dysfunction. Moreover, mice transplanted with wildtype BMC exhibit Ctns gene expression in all the tissue compartments and this expression correlates with cystine decrease measured by mass spectrometry. This work is a proof of concept for using BMC transplantation as a therapy for cystinosis. Moreover, the extensive re-population of tissue compartments highlights the efficiency of this strategy as therapy for a chronic, progressive degenerative disease.

185. Muscle Specific Gene Transfer of the PEPCK-C Improves Physical Activity in Mice

Daibang Nie,1 Dong Wei,2 Michael Y. Mi,1,4 Ying Tang,1 Allan Z. Zhao,2 Yifan Dai,3 Johnny Huard,1 Bing Wang.1 1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 2 Cell Biology & Physiology, University of Pittsburgh, Pittsburgh, PA; 3Surgery, University of Pittsburgh, Pittsburgh, PA; 4Harvard College, Harvard University, Cambridge, MA. Phosphoenolpyruvate Carboxykinase, cytosolic form (PEPCK-C) is a key enzyme involved in gluconeogenesis, glyceroneogenesis and cataplerosis (i.e. the removal of citric acid cycle anions) in liver, kidney, and adipose tissue. In humans and mammals, the low expression level of the PEPCK-C in skeletal muscle dictates that glycogen is the dominant source of energy for muscles during intensive exercise. However, this anaerobic metabolic pathway releases a small fraction of the energy contained in the glucose molecules, and the elevated concentrations of lactate in the muscle and blood cause pain in skeletal muscles (termed lactic acidosis). More recently, it was found that the overexpression of the PEPCK-C in skeletal muscle repatterned energy metabolism in transgenic mice (PEPCK-Cmus mice). These genetically engineered mice have the super-capability of running, living and breeding. All of these changes are based on the fuel-redistribution and burning off fat fuel with a high oxidative capacity in skeletal muscle during prolonged exercise to get more energy for enhanced physical activity. Recombinant adeno-associated virus (rAAV) vectors have proven to be the best gene delivery system in the muscle without immune response and toxicity compared to other viral vectors. However, it is not known if AAV vector delivering the PEPCK-C specifically in skeletal muscle will allow an increased of mitochondria and a greater contribution of intramuscular triglycerides (IMTG), and consequently to enhance running capacity. In this study, we tested our central hypothesis that highly efficient expression of the PEPCK-C specifically in skeletal muscle by AAV vector mediated systemic delivery has potential effects to enhance physical activity. We found that 1) efficient expressions of the PEPCK-C and GFP reporter genes driven by the skeletal muscle specific promoter (tMCK) were revealed in skeletal muscle one month after intraperitoneal injection (i.p.) of AAV8 vectors to neonatal C57BL/6J mice; 2) glucose tolerance and insulin tolerance testes at 10 weeks showed no remarkable difference between the treated and untreated group; 3) a weekly check of body weight did not find significant difference between the treated and untreated group; 4) comparison of the PEPCK-C treated to untreated mice at 6 and 9 weeks of age has increased endurance on treadmill test by 1.5 fold (*p < 0.005), and no gender difference was found in each group (* p > 0.05); and, 5) improvements in mitochondrial density and size were found in the PEPCK-C treated thigh muscle of 5 month-old mice using electron microscopy. Next, we will focus on evaluating the efficacy of the PEPCK-C in fatty acid oxidation in muscle, and the resultant changes in histology and physiology. The results from this study suggest a potential molecular approach for enhancing muscular Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

endurance because the PEPCK-C can alter energy metabolism by using fatty acid as a source for physical activity.

186. Bilateral Nigral rAAV5-Mediated GDNF Over-Expression Activates CRH Neurons in the Paraventricular Nucleus of the Hypothalamus and Induces Robust Weight Loss in Obese LeptinResistant Rats

Fredric P. Manfredsson,1,5 Nihal Tumer,2,3 Benedek Edros,2,3 Tessa Landa,2,3 Chritopher S. Broxson,2,3 Layla F. Sullivan,1,5 Aaron C. Rising,1,5 Kevin D. Foust,1,5 Yi Zhang,2,3 Nicholas Muzyczka,4,5 Oleg S. Gorbatuk,4,5 Philip J. Scarpace,2,3 Ronald J. Mandel.1,5 1 Department of Neuroscience, University of Florida, Gainesville, FL; 2Geriatric Researh, Department of Veterans Affairs Medical Center, Gainesville, FL; 3Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL; 4Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL; 5Powell Gene Therapy Center, University of Floirda, Gainesville, FL. Glial cell-line derived neurotrophic factor (GDNF), a member of the transforming growth factor- β family, has previously been shown to reduce body weight when delivered intracerebroventricularly in Parkinson disease patients. Additionally, our lab has shown overexpression of GDNF by recombinant adeno-associated virus (rAAV) delivered to the hypothalamus (HYP) produced weight loss in rats. These observations combined with fact that the nigrostriatal tract is known to effect feeding and weight loss, led our lab to examine the affects of injections of rAAV-GDNF into the HYP and the substantial nigra (SN) in aged F344xBN rats. The rats showed significant weight loss from the injections into both HYP and the SN compared to rAAV-GFP control injections. However, the extent of weight loss was much greater in the SN-GDNF injected animals compared to the HYP-GDNF injected animals. SN-GDNF rats showed a significant decrease in food consumptions out to 5 weeks after injection. There was no difference in activity between the groups. Hypothalamic double staining of oxytocin and phosphorylated extra-cellular signalregulated kinase (p-ERK) from SN injected was seen surrounding the area of ERK activation and indicates ERK is activated in the medial parvocellullar division (MPD) of the para-ventricular hypothalamus (PVH) because expression of the oxytocin is limited to the parvocellullar PVH. The MPD contains almost exclusively corticotrophin releasing hormone neuroendrocrine neurons, that are activated by p-ERK. GDNF over-expression in the SN may act on the noradrenergic efferrents projecting to the hypothalamus causing phosphorylation of ERK in the MPD and lead to the weight loss observed in the rats.

187.

Liver Cell Therapy from Cadaveric Donors

Laura Erker,1 Hisaya Azuma,1 Laura Eaton,1 Eric Benedetti,1 Bryan Jensen,1 Milton Finegold,2 Holger Willenbring.3 1 Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR; 2Department of Pathology, Texas Children’s Hospital, Houston, TX; 3Institute for Regeneration Medicine, University of California, San Francisco, San Francisco, CA. Due to the difficulty in acquiring whole donor livers more than 60% of patients needing a life-saving organ transplantation will not receive one. A viable alternative for some patients could be hepatocyte transplantation, but the donor cells are acquired from the same sources as whole organs and thus the supply is severely limited. Here we investigate a new, more plentiful, source of viable hepatocytes for this purpose, cells from cadaveric livers. While it is well known that whole cadaveric livers are unsuitable for transplantation, it has never been determined whether transplantable cells could be procured from such a source. In order to investigate this possibility we isolated cells from S73