401 Electric Fields Direct the Migration of Embryonic Stem Cells Derived Motor Neurons Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy S152[.]
NEUROLOGICAL DISEASES I transcriptional upregulation of cytokines and their signaling components suggesting an inflammatory component to the pain observed in the model Therefore we used a replication-defective HSV vector expressing the soluble form of the TNFα receptor (TNFαsR) that has been shown to be efficacious in spinal cord injury and bladder pain models of neuropathic pain Injection of TNFαsR expressing vector into the hindpaws of VZV sensitized rats at 21d post VZV inoculation resulted in an abrupt and prolonged (out to weeks) abrogation of mechanical (assessed using von Frey hair methodologies) and thermal hypersensitivity (using Hargreave’s apparatus) when compared to that of control vector inoculated animals These results suggest that the rat VZV-induced pain model has a strong inflammatory component that contributes to nocifensive behaviors This supports the potential of this approach alone, or in combination with enkephalin vectors as a viable therapy to treat human PHN patients 399 Negative Modulation of the Vanillloid Receptor (TRPV1) by Protein Phosphatase -1α (PP1α) Inhibits Heat- and Capsaicin-Induced but Not Cold- or Formalin-Induced Nocifensive Behaviors William F Goins,1 Bonnie Reinhart,1 Asaff Harel,1 Mingdi Zhang,1 James R Goss,1 Justus B Cohen,1 Joseph C Glorioso.1 Microbiology & Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA Chronic pain represents a major cause of morbidity, significantly impairing quality of life and imposing a substantial financial and healthcare burden The wide distribution of a limited range of neurotransmitters, receptors, and ion channels in the nervous system makes it difficult to selectively target pain-related pathways using drugs that are administered systemically and as a result tolerance, abuse and deleterious side-effects limit the use of all available therapeutics In an effort to find new pain modulatory genes, we previously used HSV-based vectors to select for gene products specified by a PC12 cell-derived cDNA library that negatively regulate TRPV1 The screen identified protein phosphatase 1α (PP1α) as a novel antagonist of capsaicin-induced TRPV1 activity, similar to the dominant-negative “poreless” TRPV1 control Expression of these products from a replication-defective HSV vector demonstrated reduced capsaicin-induced calcium influx in primary rat dorsal root ganglion (DRG) neurons in vitro compared to eGFP control vector In vivo studies in Sprague-Dawley rats showed that expression of PP1α led to increased paw withdrawal latency times using a Hargreaves apparatus, and decreased response to heat- and capsaicin-induced nocifensive behaviors in the heat ramp assay at temperatures >42oC, consistent with acting through TRPV1 These behaviors were similar to those observed with the positive control poreless TRPV1 vector but were not observed with the negative control eGFP virus Expression of PP1α or poreless TRPV1 from the vector did not alter either formalin-induced responses or menthol/icilin-induced cold behaviors, suggesting that the observed activity of PP1α is specific for TRPV1 C-fibers, but not TRPA1 (formalin-sensitive) or TRPM8 (menthol/ cold-sensitive) fibers While it remains to be determined whether PP1α phosphatase activity acts directly on TRPV1 or indirectly by affecting other modulators of TRPV1, preliminary data from PP1αtransduced DRG neurons in vitro suggest that HSV vector-mediated PP1α expression results in a decrease in pERK Together, these data demonstrate the power of our virus-based screen to identify negative modulators of TRPV1 and suggest that PP1α may be applicable as a novel agent for specific nociceptive pain control S152 400 Selective In Vivo Silencing in Astrocytes Aurélie Delzor,1,2 Nicolas Merienne,3 Amaya Viret,3 Maria Rey,3 Virginie Zimmer,3 Noëlle Dufour,1,2 Fanny Petit,1,2 Marion Chaigneau,1,2 Martine Guillermier,1,2 Diane Houitte,1,2 Emmanuel Brouillet,1,2 Philippe Hantraye,1,2 Nicole Déglon.3 Institute of Biomedical Imaging (I2BM) and Molecular Imaging Research Center (MIRCen), CEA, Fontenay-aux-Roses, France; CNRS, URA2210, Fontenay-aux-Roses, France; 3Department of Clinical Neurosciences (DNC), Laboratory of Cellular and Molecular Neurotherapies (LNCM), Lausanne University Hospital (CHUV), Lausanne, Switzerland Astrocytes represent the most abundant cell type of the brain and are indispensable for neuronal function They are implicated in neuronal dysfunction and death observed in many neurodegenerative diseases, thus making them potential targets for gene delivery systems Among these systems, viral vectors and especially lentiviral vectors (LV) offer the possibility to overexpress a transgene in the CNS We have previously developed an LV targeting specifically astrocytes by combining a Mokola G (MOK/LV) pseudotyping and miRNA detargeting (miRT) strategy (Colin et al 2009) However, this system is not suitable for astrocyte-specific silencing due to the maturation and cleavage of miR-embedded shRNA To overcome this limitation, we combined four strategies: pseudotyping of LV with Mokola G protein (MOK/LV), expression of miRNA embedded shRNA under a Tetracycline promoter, regulation of the transactivator using astrocyte specific glutamine synthetase promoter (GS) and miRT detargeting The efficacy of a vector encoding an shRNA targeting the GFP was evaluated in BAC GLT1-GFP transgenic mice expressing the reporter gene exclusively in astrocytes To demonstrate the specificity of the silencing, we used a bicistronic LV, expressing GFP and mCherry into neurons The Cherry/GFP ratio was used to quantify potential residual silencing in neurons Our results demonstrate that astrocyte-specific gene silencing can be achieved by an inducible system regulated by an astrocytic promoter and miRT detargeting We are currently using this astrocytic LV to specifically silence mutant huntingtin in astrocytes in order to evaluate the contribution of astrocytes in HD and the need to target these cells to alter the progression of the pathology 401 Electric Fields Direct the Migration of Embryonic Stem Cells Derived Motor Neurons Yongchao Li,1 Mark Weiss,2 Li Yao.1 Department of Biological Sciences, Wichita State University, Wichita; 2Kansas State University, Manhattan Spinal cord injury or diseases such as amyotrophic lateral sclerosis can cause the loss of motor neurons and therefore result in muscular paralysis Stem cells may improve functional recovery after injury by promoting endogenous regeneration, or directly replacing neurons Effective directional migration of grafted neural cells to reconstruct functional connections is crucial in the process Steady direct current electric fields (EFs) play an important role in the development of the central nervous system A strong biological effect of EFs is the induction of directional cell migration In this study, embryonic stem (ES) cells were differentiated using a two-step procedure The first step was to enhance the neuralization process by adding Noggin and fibroblast growth factor in the cell culture medium The second step was to induce motor neuron specification by adding retinoic acid and smoothened agonist as cell culture culture supplement The cells were then treated with brain-derived neurotrophic factor, ciliary neurotrophic factor, glial cell-derived neurotrophic factor and neurotrophin-3 for neurite elongation The migration of neural precursors from embryoid bodies in EFs was studied We found that the migration of neural precursors from the embryoid body was toward the cathode pole We also investigated the EFs-directed migration of dissociated single motor neurons using time-lapse photography Similarly, single motor neurons migrated to the cathode and the Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy MUSCULOSKELETAL CELL AND GENE THERAPY reversal of EFs poles reversed the migration direction of the cells The directedness and displacement of cathodal migration became more significant when the field strength was increased from 50mV/mm to 100mV/mm EFs stimulation did not influence the cell migration velocity Our work suggested that EFs may serve as a guidance cue to direct grafted cell migration in vivo Musculoskeletal Cell and Gene Therapy 402 Gait Function in a Canine Model of X-Linked Myotubular Myopathy After Systemic Delivery of AAV8-MTM1 Melissa A Goddard,1,2 David L Mack,2,3 Jessica M Snyder,4 Michael W Lawlor,5 Alan H Beggs,6 Anna Buj-Bello,7 Martin K Childers.2,3 Department of Physiology and Pharmacology, Wake Forest University, Winston Salem, NC; 2Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA; Department of Rehabilitation Medicine, School of Medicine, University of Washington, Seattle, WA; 4Department of Comparative Medicine, University of Washington, Seattle, WA; Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, Milwaukee, WI; 6Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children’s Hospital, Harvard Medical School, Boston, MA X-linked myotubular myopathy (XLMTM), the most common of the centronuclear myopathies, is caused by lack of myotubularin due to mutation of the MTM1 gene Affected boys experience profound weakness of the skeletal muscles and most are ventilator- and wheelchair-dependent Although XLMTM patients are typically non-ambulatory, spatio-temporal changes in gait like speed and stride length are well-established to quantify changes in muscle and limb function in canine models of neuromuscular disorders These can also be indicative of the impact of the disease on quality of life, which is of critical importance to patients and their families We assessed gait in the XLMTM dog model, which is MTM1-null and displays a phenotype similar to that seen in humans At 10 weeks of age, an adeno-associated virus, serotype (AAV8) encoding a full-length canine MTM1 cDNA was infused into the cephalic vein of XLMTM dogs Dogs were given either a “low-dose” (5 x 10 12 vg/kg) (n=3) or a “high-dose (2.5 x 10 13 vg/kg) (n=3), with the remaining dogs receiving a saline control (n=4) XLMTM dogs (n=10) and their normal true littermates (n=5) were measured before dosing, every weeks until 21 weeks of age and every weeks thereafter until 41 weeks of age Dosing and measurement was carried out in a blinded fashion To assess gait, dogs walked along an instrumented carpet at a self-selected pace and software determined speed and stride length XLMTM dogs that received saline had greatly reduced speeds and much shorter strides than wildtype littermates However, dogs given high-dose AAV8 have spatio-temporal measures that approach wildtype values after treatment, suggesting that AAV8-MTM1 replacement improves limb function Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy Figure 1: Gait speed over time as measured by the instrumented carpet 403 Improved Respiratory Function Is Associated With Increased Survival After Systemic Delivery of AAV8-MTM1 in a Canine Model of X-Linked Myotubular Myopathy Melissa A Goddard,1,2 David L Mack,2,3 Jessica M Snyder,4 Robert W Grange,5 Michael W Lawlor,6 Alan H Beggs,7 Anna Buj-Bello,8 Martin K Childers.2,3 Department of Physiology and Pharmacology, Wake Forest University, Winston-Salem, NC; 2Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA; Department of Rehabilitation Medicine, School of Medicine, University of Washington, Seattle, WA; 4Department of Comparative Medicine, University of Washington, Seattle, WA; Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic and State University, Blacksburg, VA Absence of the protein, myotubularin encoded by the MTM1 gene, causes X-linked myotubular myopathy (XLMTM), a fatal pediatric disease estimated to impact in 50 000 male births Affected boys have severe muscle weakness including the respiratory muscles; most patients require ventilatory support Respiratory insufficiency is the leading cause of death There is currently no treatment but therapies under development aim to replace the missing gene or gene product A naturally-occuring canine MTM1 mutation provides a model of the disease in dogs with a clinical presentation similar to patients We hypothesize that respiratory muscle weakness due by the deficiency negatively impacts survival by impairing function XLMTM dogs (n=10) were infused via the cephalic vein at 10 weeks of age and received either saline (n=4) or an adeno-associated virus, serotype (AAV8) encoding a full-length canine MTM1 cDNA, at two doses: a “low-dose” (5 x 1012 vg/kg) (n=3) or a “high-dose (2.5 x 1013 vg/kg) (n=3) Animals were assessed in a blinded fashion and compared to true littermate normal controls (n=6) prior to treatment, every weeks until 21 weeks of age and then every weeks until 41 weeks of age Doxapram chloride was administered to stimulate respiration and the peak inspiratory flow (PIF) was measured using a pneumotach While wildtype dogs maintain PIF over time, reduction in the PIF of the untreated XLMTM dogs is associated with an abbreviated lifespan PIF for dogs receiving low dose AAV8-MTM1 remains subnormal but increases in dogs given high dose treatment We conclude that maintained respiratory function contributes to continual survival, which may be preserved by high-dose AAV8-MTM1 treatment S153 ...MUSCULOSKELETAL CELL AND GENE THERAPY reversal of EFs poles reversed the migration direction of the cells The directedness and displacement of cathodal migration became more significant when the field strength... models of neuromuscular disorders These can also be indicative of the impact of the disease on quality of life, which is of critical importance to patients and their families We assessed gait in the. .. myopathy (XLMTM), the most common of the centronuclear myopathies, is caused by lack of myotubularin due to mutation of the MTM1 gene Affected boys experience profound weakness of the skeletal muscles