403 Improved Respiratory Function Is Associated With Increased Survival After Systemic Delivery of AAV8 MTM1 in a Canine Model of X Linked Myotubular Myopathy Molecular Therapy Volume 22, Supplement 1[.]
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 injection Similar effects with 2-fold increase were recapitulated when IL-10-transduced MSCs were injected into dog, without elevation of serum levels of IL-10 The accumulation of MSCs was also found at the site of CTX-injured muscle at weeks after the intra-arterial injection into the dog Furthermore, the long-term engraftment permitted MSCs to form muscle fibers without any stimulation or treatment for myogenic differentiation Conclusion: Various beneficial effects on engraftment were observed when MSCs were engineered to express IL-10 The strategy to use IL-10-producing MSCs would be promising for the anti-inflammatory therapy of muscle diseases including DMD due to their long-term engraftment and survival, although further study is required for understanding the mechanism of the process 405 Hindlimb and Forelimb Strength Assessment in a Canine Model of X-Linked Myotubular Myopathy Following AAV-Mediated Gene Replacement Figure 1: Changes in respiratory function over time for untreated and treated XLMTM dogs and their normal true littermates 404 Skeletal Muscle Engraftment of Mesenchymal Stromal Cells Is Augmented By IL-10 Yuko N Kasahara,1 Hiromi H Kinoh,1 Tomoko Chiyo,1 Hironori Okada,1 Shin’ichi Takeda,1 Takashi Okada.1 Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan Background: Multipotent mesenchymal stromal cells (MSCs) are potential therapeutics because of their ability to differentiate into various cell types in situ with immunosuppressive properties and have been approved for the treatment of inflammatory diseases We previously reported a strategy to expand canine MSC cultures with myogenic differentiation, which is promising for the treatment of Duchenne muscular dystrophy (DMD) However, further development of these approaches are required to obtain functional recovery of the skeletal muscles in DMD patients Because anti-inflammatory IL-10 has anti-apoptotic properties, we hypothesized that increased IL-10 production in MSCs should improve the limited survival of the transplanted MSCs to enhance the immunosuppressive function of MSCs We investigated the effects of IL-10 on the MSCs engraftment in the skeletal muscle for future DMD therapy with MSCs Methods: (1) Rat MSCs expressing luciferase (Luc-MSCs, 5-7.5x106 cells) with recombinant IL-10 (1μg) or IL-10-expressing rAAV-1 vector (5-10x108 g.c.), (2) IL-10 plasmids-trasnfected LucMSCs, or (3) IL-10/rAAV-1 vector-transduced Luc-MSCs were intramuscularly injected into NOD/SCID mice After the injection, in vivo luminescence imaging was periodically performed to assess engraftment efficiency and cell survival in the transplanted mice Canine CD271-positive MSCs obtained from bone marrow were transduced with IL-10/rAAV-1 (5x108 cells) and then were administered into the cardiotoxin (CTX)-injured TA muscle or the femoral artery of Beagle dogs The transplanted muscles were histologically analyzed Results: (1) When recombinant IL-10 was repeatedly injected, the retention of Luc-MSCs was slightly increased at 2-4 days after the injection as MSCs without IL-10 The MSCs survival was similar in both cases of MSCs administration with or without IL-10/AAV-1 vector, suggesting that IL-10 level was not sufficient to regulate cell death in the muscle at days after the injection (2) IL-10-trasnfected MSCs, which were expressing IL-10 could be efficiently engrafted in muscle (69-fold higher signal intensity of luciferase) compared with untransfected cells (3) IL-10/AAV-1 vector-transduced Luc-MSCs showed significant increase of engraftment (9-fold) when compared with untransduced MSCs and survived for more than weeks after the S154 Stefan Czerniecki,1,2 Robert W Grange,3 Jon A Doering,3 JeanYves Hogrel,4 David L Mack,1,2 Anna Buj-Bello,5 Martin K Childers.1,2 Department of Rehabilitation Medicine, University of Washington, Seattle, WA; 2Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA; 3Department of Human Nutrition, Foods and Exercise, Virginia Polytechnic and State University, Blacksburg, VA; 4Institut de Myologie, Paris, France X-linked myotubular myopathy (XLMTM), a centronuclear myopathy caused by a mutation in the MTM1 gene, leads to an absence of functional myotubularin, a lipid phosphatase Affected patients display profound muscle weakness that leads to loss of ambulation and in most cases, respiratory failure We recently described the use of an adeno-associated virus based gene therapy (AAV8-MTM1) in a naturally occurring canine model of this disease A single intravascular administration extended the survival and markedly improved hindlimb muscle strength in these dogs In the present study, we evaluated strength in both hindlimb and forelimb muscles Percutaneous stimulation of peripheral nerves in the hind- and forelimb leads to muscular contraction Resulting force (torque) of contraction is measured using a pedal connected to a custom transducer/ergometer, and normalized to the animals’ body mass We used linear regression to determine if the effects of AAV gene therapy yielded similar parallel improvements in both fore and hindlimb muscle force, with the idea that measures in the forelimb might predict hindlimb strength, or vice versa Results (Figure 1) show good correlation (R= 0.86; R2 = 0.74) between forelimb extension and hindlimb flexion max torque in the affected animals treated with a high dose of AAV8-MTM1 (2.5x1013 vg/kg), while the carrier controls showed only a modest correlation (R= 0.69; R2 = 0.48) Animals treated with a low dose of the gene therapy (5x1012 vg/kg), however, showed a weak correlation between forelimb extension, and hindlimb flexion max torque (R= 0.49; R2 = 0.24, slope not significantly different than 0) Untreated affected animals showed a modest correlation (R=0.64; R2 = 0.41) between the same measurements, which manifests as a progressive loss of muscle strength as the animals age These findings support the conclusion of a dose-dependent effect of AAV therapy in this model, and demonstrate the high dose AAV-MTM1 leads to a parallel strength gain in both forelimb and hindlimb musculature The predictive utility of the regression for the high dose treatment remains to be tested Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... adeno -associated virus based gene therapy (AAV8- MTM1) in a naturally occurring canine model of this disease A single intravascular administration extended the survival and markedly improved hindlimb... Blacksburg, VA; 4Institut de Myologie, Paris, France X- linked myotubular myopathy (XLMTM), a centronuclear myopathy caused by a mutation in the MTM1 gene, leads to an absence of functional myotubularin,... understanding the mechanism of the process 405 Hindlimb and Forelimb Strength Assessment in a Canine Model of X- Linked Myotubular Myopathy Following AAV-Mediated Gene Replacement Figure 1: Changes in respiratory