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458 External, Non Invasive Monitoring of Progressive Cardiorespiratory Dysfunction in a Canine Model of DMD Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene[.]
MUSCULO-SKELETAL GENE & CELL THERAPY II muscle regeneration are muscle stem cells and satellite cells Here in this study, transgenic myoblasts (with the Cre/Lox-β-galactosidase system) were transplanted into skeletal muscle of adult mouse model for determining the process of dedifferentiation after injury METHODS: Cre-Lox system: Myocytes from a normal mouse were isolated and transfected with a Cre-expressing retrovirus to generate M-Cre The M-Cre were co-cultured with another population of myocytes isolated from 129-Gt(ROSA)26Sortm1Sho (Lox-βgeo) reporter mice (M-Lox) Fusion of M-Cre and M-Lox resulted in Cre recombinase-mediated cleavage of Lox sites and induction of β-gal expression in resulting fused myotubes Muscle injury: weeks after transplantation of Cre-Lox cells into gastrocnemius (GM) muscle of SCID mice (C57BL/6J), lacerations were performed GM of one leg, and the GM muscle of the other leg serves as control Isolation of muscle cells: days after muscle injury, the cells were isolated with pre-plate technique The isolated cells were cultured in the proliferation medium [DMEM supplemented with 10% Fetal Bovine Serum (FBS), 10% Horse Serum (HS), 1% Penicillin-Streptomycin antibiotics, and 0.5% chicken essential extract (CEE)] RESULTS: The function of Cre-Lox system in differentiated myotubes in vitro: days after myogenic differentiation, β-gal expression was exclusively observed in multinuclear myotubes in the mixed culture of Cre-cells and Lox cells Cre/Lox-β-galactosidase cell transplantation and follow up muscle injury resulted in β-gal positive mononucleated cells, indicating the occurring of muscle cell dedifferentiation β-galactosidase positive cells from injured muscle stay in different populations (myoblasts, satellite cells, and muscle stem cells) DISCUSSION: Our results demonstrate that dedifferentiation may occur in injured skeletal muscle The process of dedifferentiation in the differentiated muscle cells is proved by our Cre-Lox system Mononuclear cells released from differentiated (β-galactosidase positive) myocytes, which is a single population of cells at beginning, were shown to have developed into different cell populations including MyoD+ myoblasts, Pax-7+ satellite cells, and Sca-1+/CD34+ muscle stem cells Therefore, the dedifferentiation process may contribute to muscle regeneration during muscle injury and repair Acknowledgement: Authors acknowledge funding from NIH and DOD 457 Increased Muscle Regeneration in Hindlimb and Diaphragm of mdx Mice Treated with AAV9 Mini-Dystrophin and Octalysine-NEMO Binding Domain Peptide Daniel P Reay,1 Geno Raggi,1 Bing Wang,2 Xiao Xiao,5 Paul D Robbins,3 Paula R Clemens.1,4 Neurology, University of Pittsburgh, Pittsburgh, PA; 2Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; 3Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA; 4Department of Veteran’s Affairs, Pittsburgh, PA; 5School of Pharmacy, University of North Carolina, Chapel Hill, NC The loss of a functional, membrane-localized dystrophin protein is the primary cause of Duchenne muscular dystrophy (DMD) The muscle inammatory inltrates and failure of muscle regeneration that are secondary to dystrophin loss have recently been shown to be due, in part, to activation of the nuclear factor κB (NF-κB) Dystrophic muscle shows substantially increased nuclear accumulation of NFκB, which is well known to regulate cytokines, other inammatory molecules and myogenic proteins Toward the development of therapy for DMD, we designed an experiment to test the combination of dystrophin gene replacement and inhibition of NF-κB activation For dystrophin gene replacement therapy, we utilized an AAV9 vector to systemically deliver a mini-dystrophin transgene to day old neonatal dystrophin-decient mdx mice Beginning at weeks of age, mice were then treated with tri-weekly injections of octalysine-NEMO binding domain peptide (8K-NBD), to inhibit Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy NF-κB activation Mice treated with either AAV9 mini-dystrophin vector alone or AAV9 mini-dystrophin vector plus 8K-NBD peptide exhibited mini-dystrophin expression and reduced necrosis in the quadriceps and diaphragm, but mice treated with the AAV9 minidystrophin vector plus 8K-NBD peptide additionally demonstrated increased levels of muscle regeneration in quadriceps and diaphragm EMSA analysis for NF-κB conrmed decreased levels of nuclear NF-κB in 8K-NBD-treated mdx mice Our data suggest that high levels of mini-dystrophin gene transfer are required to demonstrate a reduction in the nuclear accumulation of NF-κB in muscle of mdx mice treated with vector alone Overall, treatment of mdx mice with AAV9 mini-dystrophin gene replacement therapy in combination with NBD peptide/NF-κB inhibitory therapy may provide critical insight into potential treatments for DMD 458 External, Non-Invasive Monitoring of Progressive Cardiorespiratory Dysfunction in a Canine Model of DMD Andrew Mead,1 Alock Malik,1 Mihail Petrov,1 Martin Childers,2 Janet Bogan,3 Joseph Kornegay,3 Hansell Stedman.1 University of Pennsylvania, Philadelphia, PA; 2Wake Forest University, Winston-Salem, NC; 3University of North Carolina, Chapel Hill, NC The GRMD (Golden Retriever Muscular Dystrophy) model for Duchenne Muscular Dystrophy (DMD) mimics human disease progression with regard to both histopathology and locomotive function However, unlike DMD, the GRMD model exhibits a high degree of variability in both the severity and speed of progression, even among littermates This aspect of the model poses challenges for assessing efficacy in pre-clinical therapeutic trials of gene therapy, and requires further improvement in quantitative measures of disease progression Electrical stimulation of distal limb muscles has provided the most reliable quantitative data, however this requires serial episodes of general anesthesia, with attendant risks of cardiopulmonary complication in the dystrophic dogs Moreover, the data are limited to locomotive muscle function Most non-invasive means of assessment, such as running times, are unreliable due to their volitional nature, and fail to dissect the cardiac, respiratory and locomotive components of disease progression Here we investigate the use of a non-invasive device that records EKG, 3D accelerometry, and 2-band plethysmography in a wireless package that has negligible effect on animal behavior, the “Lifeshirt” (Vivometrics) In particular we simultaneously examine cardiac and respiratory function, two physiological parameters relevant to longevity in DMD We hypothesize that the progressive loss of ventilatory reserve caused by degeneration of the diaphragm will manifest, in the GRMD model, as a progressive respiratory paradox, (i.e asynchrony in the chest wall and abdominal wall excursion) during and immediately after mild exertion The degree of paradox will correlate with trans-diaphragmatic pressure development during stimulated spontaneous breathing and phrenic nerve stimulation at the time of necropsy Progressive heart failure, also a hallmark of DMD in humans, will manifest rst as parasympathetic withdrawal measured by depressed respiratory sinus arrhythmia in affected dogs as compared to unaffected littermates Decreased RSA would be expected to correlate with impaired cardiac contractility as measured by more direct means, e.g Langendorff isolated perfused heart studies performed post mortem Data obtained using the “Lifeshirt”system on GRMD pups reveals that by months of age there are early indicators of respiratory and cardiac dysfunction In preliminary studies of 11 dystrophic and normal dogs under months of age RSA was signicantly reduced in the affected group (p