184 Successful Treatment of Cystinosis Using Bone Marrow Cell Transplantation Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S72 METABOLIC DISEASE[.]
METABOLIC DISEASES I isolated from PKGI-deficient mice showed reduced levels of TGs, brown adipogenic markers and mitochondria (as assessed by RQPCR of mitochondrial DNA encoded genes) indicating an arrest of BAT development at an immature stage Furthermore, thermogenic activity of the mutant new born mice was reduced, notably in the neck region, as detected by thermographic imaging Taken together, these data show a so far unknown role for PKGI in mesenchymal stem cell differentiation and implicate a new cGMP-dependent pathway in the differentiation process of MSCs Given the recent findings that metabolically active BAT is also present in humans adults, regulation of BAT differentiation by NO/cGMP might have clinical relevance for the treatment of obesity and metabolic disorders 182 A Method To Determine the In Vivo Oxidative Capacity for 13C Isotopomers in Mice: Use To Study Intermediary Metabolism and To Monitor Transgene Activity Charles P Venditti,1 Eirini Manoli,1 Randy J Chandler.1 GMBB/NHGRI/NIH, Bethesda, MD Background: A number of amino, organic, and fatty acid oxidation pathways feature the sequential generation of carbon dioxide as the carbon skeletons derived from substrate precursors flow toward oxidation The use of 13C isotopomers coupled with determination of CO2 production can be incorporated into the analysis of substrate oxidation in a given metabolic pathway by measuring the enrichment of 13CO2 produced after oral or parenteral delivery of the stable isotope The monitoring of pathway activity in this fashion might allow non-radioactive, repeatable, in vivo determinations of whole body metabolism to be easily and precisely made on single mice Methods: We have designed and constructed closed circuit, constant volume respiratory chambers for the collection and quantitation of 13CO2 from mice The chambers have removal and addition ports to allow sampling of gas and are fitted with a CO2 probe and an O2 sensor to allow real time measurement of expired CO2 and O2 consumption To determine substrate oxidation, an animal is injected with a tracer dose of a 13C substrate precursor, placed in the chamber and aliquots of expired air are then sequentially removed for analysis of 13CO2 enrichment using isotope ratio mass spectroscopy (IRMS) Together, the simultaneous measurement of enrichment and CO2 production by the animal under study allows the oxidation of the tracer to be directly measured Results: We have used this method to examine the oxidation of 13C-isotopomers of propionate, valine, leucine, phenylalanine, glycine, and methionine in control and methylmalonyl-CoA mutase (Mut) knock-out mice The mutant animals can metabolize 13C-leucine and 13C-valine as well as controls, indicating that the early steps of mitochondrial branched chain aminoacid oxidation is intact, but show a highly reproducible defect in the ability to oxidize 13C-propionate into 13CO2 Furthermore, secondary metabolic perturbations in the oxidation of 13C-glycine and 13C-methione were detected in the mutants, even though the circulating metabolite pools for these aminoacids were present in the same concentration as in control littermates As an extension of this technique, Mut mice that either received rAAV gene therapy (Abstract 950387) or harbored tissue specific transgenes were studied The gene therapy treated animals and knock-out mice that expressed the Mut gene at a low level from a liver specific transgene had significantly increased 13C-propionate oxidation, despite a large dilution effect of the label due to an expanded tracee pool size Conclusions: 13CO2 generation from labeled isotopomers using this newly developed method has provided: new insights and means to monitor the secondary perturbations of amino acid metabolism in methylmalonic acidemia A robust technique to assess the in vivo metabolic effects of transgene expression, delivered by gene therapy vectors or through the germline The method should be broadly extendable to study other disorders of amino, organic and fatty acid S72 metabolism and practically useful to repeatedly and non-invasively quantitate enzyme and pathway activity, in mice and patients 183 Erythropoietin Over-Expression Protects Against Diet-Induced Obesity through Increase of Fat Oxidation in Muscle in Mice Pernille Hojman,1 Hanne Gissel,2 Claus Brandt,1 Bente Klarlund Pedersen,1 Julie Gehl.3 Center for Inflammation and Metabolism, Unversity of Copenhagen, Copenhagen, Denmark; 2Department of Physiology and Biophysics, University of Aarhus, Aarhus, Denmark; Department of Oncology, University of Copenhagen, Herlev, Denmark Erythropoietin can be over-expressed in skeletal muscles by gene electrotransfer, resulting in 100-fold increase in serum EPO and significant increases in haemoglobin levels Earlier studies have suggested that EPO improves several metabolic parameters when administered to chronically ill kidney patients Thus we applied the EPO over-expression model to investigate the mechanisms whereby EPO mediates its metabolic roles At 12 weeks, EPO expression resulted in a 23% weight reduction (P < 0.01) in EPO transfected obese mice; thus the mice weighed 21.9 ± 0.8 g (control, normal diet,) 21.9 ± 1.4 g (EPO, normal diet), 35.3 ± 3.3 g (control, high-fat diet) and 28.8 ± 2.6 g (EPO, high-fat diet) Correspondingly, DXA scanning revealed that the reduced body weight was due to a 28% reduction in adipose tissue mass The decrease in adipose tissue mass was accompanied by a complete normalisation of fasting insulin levels and insulin sensitivity in the high-fat fed mice EPO expression also induced a 14% increase in muscle volume and a 25% increase in vascularisation of the EPO transfected muscle Muscle force and stamina were not affected by EPO expression PCR array analysis revealed that genes involved in lipid metabolism, thermogenesis and inflammation were increased in muscles in response to EPO expression, while genes involved in glucose metabolism were down-regulated.In conclusion, we show that EPO has substantial metabolic effects including protection against diet-induced obesity and normalisation of glucose sensitivity associated with a shift to increased fat metabolism in muscle 184 Successful Treatment of Cystinosis Using Bone Marrow Cell Transplantation Kimberly Syres,1 Frank Harrison,1 James V Jester,2 Jennifer Simpson,2 Subhojit Roy,3 Daniel R Salomon,1 Stephanie Cherqui.1 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA; 2Department of Ophthalmology, University of California Irvine, Orange, CA; Department of Neurosciences, University of California San Diego, La Jolla, CA Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders Cystinosis results from a genetic defect in the gene CTNS encoding the lysosomal cystine transporter protein, cystinosin Cystine accumulates in every organ in the body and leads to organ damage and dysfunction including renal defects Using the murine model for cystinosis, Ctns-/- mice, we performed syngeneic bone marrow cell (BMC), hematopoietic stem cells (HSC) and mesenchymal stem cell (MSC) transplantation Organ-specific cystine content was reduced by 57% to 94% in all organs tested in the BMC or HSC-treated mice Confocal microscopy and quantitative-PCR revealed a large quantity of transplanted BMC in all organs tested, from 5% to 19% of the total cells Most of these cells were not from the lymphoid lineage, but part of the intrinsic structure of the organ The natural progression of renal dysfunction was prevented and deposition of corneal cystine crystals was significantly improved in the BMC-treated mice In contrast, MSC did Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy 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 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 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 and 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 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 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 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 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 ... 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,... 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... Mandel.1,5 Department of Neuroscience, University of Florida, Gainesville, FL; 2Geriatric Researh, Department of Veterans Affairs Medical Center, Gainesville, FL; 3Department of Pharmacology and