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467. Detailed Physical Mapping of Deletion Breakpoints in the GSHP Dog Model for DMD
MUSCULO-SKELETAL GENE & CELL THERAPY II isolated from the skeletal muscle of mice (C57BL/6J) in lacerationinjured or non-injured status with the preplate technique. Cells were cultured in the growth medium for stem cells [DMEM supplemented with 20% Fetal Bovine Serum (FBS), 10% Horse Serum (HS), 1% Penicillin-Streptomycin antibiotics, and 0.5% chicken embryo extract (CEE)], and incubated in 5% CO2 at 37 °C. RESULTS: 1. In vivo, the activation and proliferation of muscle stem cells in the laceration-injured muscle (4 days after injury) was greatly improved compared to normal muscle. 2. MDSCs isolated from injured muscle showed improved proliferation and migration capacities, compared to MDSCs from normal muscle. 3. MDSCs from injured muscle contain more Sca-1+/ CD34+ cells, compared to that of normal muscle. 4. MDSCs from injured muscle demonstrated stronger myogenic and tissue regenerative capacity both in vitro and in vivo. 5. MDSCs from injured muscle seem to have some limited characteristics similar to pluripotent stem cells, with the activation STAT3 and NOTCH1 on mRNA level. DISCUSSION: This study took advantage of the efcient pre-plate technique and isolated a population of slow adhering stem cells which contains a larger population of Sca-1+/ CD34+ stem-cell like cells compared to those from normal muscle. Our results have proven that slow adhering stem cells from injured muscle demonstrated improved migration ability, proliferation rate, myogenic capacity, and was able to effectively repair dystrophic muscle in MDX mice. Our results can be a conrmative evidence for isolating highly regenerative stem cells from injured muscle to be applied in stem-cell based therapies, and further veries a potential value of the stem cells from injured muscle in improving the regeneration of multiple tissues. Acknowledgement: Authors acknowledge funding from NIH and DOD.
466. Sarcolemmal nNOS Anchoring Reveals a Functional Difference between Dystrophin and Utrophin
Yongping Yue,1 Dejia Li,1 Luke Judge,2 Akshay Bareja,3 Yi Lai,1 Kay E. Davies,3 Jeffrey S. Chamberlain,2 Dongsheng Duan.1 1 Department of Molecular Microbiology and Immunology, The University of Missouri, Columbia, MO; 2Department of Neurology, The University of Washington, Seattle, WA; 3Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deciency. In normal muscle, dystrophin help maintain sarcolemmal stability by linking the extracellular matrix and the cytoskeleton. Further, dystrophin recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Reduced sarcolemmal integrity has been considered as a major pathogenic mechanism in DMD. Interestingly, recent studies suggest that failure to anchor nNOS to the membrane also contributes to muscle fatigue. Over the last two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence homology, structural similarity and functional properties with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. The full-length utrophin cDNA was expressed in dystrophin-decient mdx mice by gutted adenovirus or via transgenic over-expression. Subcellular nNOS localization was determined by immunouorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supraphysiological utrophin expression, we did not detect nNOS at the sarcolemma. Our results suggest that full-length utrophin does not anchor nNOS to the sarcolemma. This nding may have important implications in developing utrophin-based DMD therapies.
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467. Detailed Physical Mapping of Deletion Breakpoints in the GSHP Dog Model for DMD
Alock Malik,1 Acong Xu,1 Jesse Chen,1 Andrew Mead,1 Martin Childers,2 Janet Bogan,3 Scott Schatzberg,4 Joe Kornegay,3 Hansell Stedman.1 1 University of Pennsylvania, Philadelphia, PA; 2Wake Forest University, Winston-Salem, NC; 3University of North Carolina, Chapel Hill; 4University of Georgia, Athens. The German Short Haired Pointer model for Duchenne muscular dystrophy (GSHPMD) may provide a unique opportunity to study the immunologically relevant aspects of gene therapy for DMD as it might provide an unmitigated dystrophin null environment unlike the other known animal models for DMD (e.g. mdx, GRMD, in which point mutations result in dystrophin deciency with the potential for reversion or read-through by alternative splicing). Although earlier studies revealed the GSHPMD mutation to be a visible deletion within the p21 region of the canine X chromosome, the precise boundaries of this deletion and their relationship to the anking genes are not yet known. In order to localize the deletion breakpoints a PCR based strategy was used. A 9Mbp region anking the canine dystrophin gene was targeted to strategically design various primers to amplify the region syntenic to the human Xp21 region. Based on the PCR amplication prole the deletion boundaries were quickly resolved to within a few thousand base pairs. Our data suggest that the deletion covers approximately 3Mbp upstream of the 5’UTR and ends very close to the 3’UTR knocking out the entire dystrophin gene and most likely affecting at least one anking gene, BCMP1, as well. We anticipate sequencing across the deletion breakpoint in the coming weeks, with the opportunity to dene the most probable genomic mechanism resulting in the deletion. Accurate description of the breakpoints of the deletion will help in early screening and carrier detection in GSHPMD colony, and will allow a precise depiction of the exons removed. The GSHPMD model may play a key translational role in the future development of therapies for DMD because of the anticipated complete absence of any dystrophin open reading frame and hence utility as a surrogate, from the standpoint of potential immunological response to recombinant dystrophin, for all DMD gene deletions found in the human gene pool. This model may also represent a unique opportunity to dene the physiological role of the highly conserved “brain cell membrane protein 1” (BCMP1), a highly expressed, brain-specic, putative four-transmembrane protein previously suggested to be a candidate gene for X-linked mental retardation.
468. Lentiviral Vector Mediated Delivery of FullLength Dystrophin for Gene Therapy of Muscular Dystrophy
En Kimura,1 Katsuhisa Uchio,1 Tomohiro Suga,1 tatsuya Koide,1 Yuji Uchida,2 Yasushi Maeda,1 Satoshi Yamoshita,1 Jeffrey S. Chamberlain,3 Makoto Uchino.1 1 Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; 2Pharmacology, Sojo University, Kumamoto, Japan; 3Neurology, University of Washington School of Medicine, Seattle, WA. Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder, and there is currently no effective treatment. DMD causes respiratory and/or cardiac failure and results in death at about 20 years of age. Lentiviral vectors are an efcient gene delivery tool for skeletal muscle bers as well as myogenic progenitor cells in vivo[/italic] and ex vivo[/italic]. The integration ability of lentiviral vectors is a huge advantage for targeting DMD myogenic cells, which require the missing gene product, dystrophin, to be expressed permanently, which can not be achieved with non-integrating vectors. We have shown that stable transduction of myogenic stem cells in Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy
466. Sarcolemmal nNOS Anchoring Reveals a Functional Difference between Dystrophin and Utrophin
Yongping Yue,1 Dejia Li,1 Luke Judge,2 Akshay Bareja,3 Yi Lai,1 Kay E. Davies,3 Jeffrey S. Chamberlain,2 Dongsheng Duan.1 1 Department of Molecular Microbiology and Immunology, The University of Missouri, Columbia, MO; 2Department of Neurology, The University of Washington, Seattle, WA; 3Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, United Kingdom. Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deciency. In normal muscle, dystrophin help maintain sarcolemmal stability by linking the extracellular matrix and the cytoskeleton. Further, dystrophin recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Reduced sarcolemmal integrity has been considered as a major pathogenic mechanism in DMD. Interestingly, recent studies suggest that failure to anchor nNOS to the membrane also contributes to muscle fatigue. Over the last two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence homology, structural similarity and functional properties with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. The full-length utrophin cDNA was expressed in dystrophin-decient mdx mice by gutted adenovirus or via transgenic over-expression. Subcellular nNOS localization was determined by immunouorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supraphysiological utrophin expression, we did not detect nNOS at the sarcolemma. Our results suggest that full-length utrophin does not anchor nNOS to the sarcolemma. This nding may have important implications in developing utrophin-based DMD therapies.
S180
467. Detailed Physical Mapping of Deletion Breakpoints in the GSHP Dog Model for DMD
Alock Malik,1 Acong Xu,1 Jesse Chen,1 Andrew Mead,1 Martin Childers,2 Janet Bogan,3 Scott Schatzberg,4 Joe Kornegay,3 Hansell Stedman.1 1 University of Pennsylvania, Philadelphia, PA; 2Wake Forest University, Winston-Salem, NC; 3University of North Carolina, Chapel Hill; 4University of Georgia, Athens. The German Short Haired Pointer model for Duchenne muscular dystrophy (GSHPMD) may provide a unique opportunity to study the immunologically relevant aspects of gene therapy for DMD as it might provide an unmitigated dystrophin null environment unlike the other known animal models for DMD (e.g. mdx, GRMD, in which point mutations result in dystrophin deciency with the potential for reversion or read-through by alternative splicing). Although earlier studies revealed the GSHPMD mutation to be a visible deletion within the p21 region of the canine X chromosome, the precise boundaries of this deletion and their relationship to the anking genes are not yet known. In order to localize the deletion breakpoints a PCR based strategy was used. A 9Mbp region anking the canine dystrophin gene was targeted to strategically design various primers to amplify the region syntenic to the human Xp21 region. Based on the PCR amplication prole the deletion boundaries were quickly resolved to within a few thousand base pairs. Our data suggest that the deletion covers approximately 3Mbp upstream of the 5’UTR and ends very close to the 3’UTR knocking out the entire dystrophin gene and most likely affecting at least one anking gene, BCMP1, as well. We anticipate sequencing across the deletion breakpoint in the coming weeks, with the opportunity to dene the most probable genomic mechanism resulting in the deletion. Accurate description of the breakpoints of the deletion will help in early screening and carrier detection in GSHPMD colony, and will allow a precise depiction of the exons removed. The GSHPMD model may play a key translational role in the future development of therapies for DMD because of the anticipated complete absence of any dystrophin open reading frame and hence utility as a surrogate, from the standpoint of potential immunological response to recombinant dystrophin, for all DMD gene deletions found in the human gene pool. This model may also represent a unique opportunity to dene the physiological role of the highly conserved “brain cell membrane protein 1” (BCMP1), a highly expressed, brain-specic, putative four-transmembrane protein previously suggested to be a candidate gene for X-linked mental retardation.
468. Lentiviral Vector Mediated Delivery of FullLength Dystrophin for Gene Therapy of Muscular Dystrophy
En Kimura,1 Katsuhisa Uchio,1 Tomohiro Suga,1 tatsuya Koide,1 Yuji Uchida,2 Yasushi Maeda,1 Satoshi Yamoshita,1 Jeffrey S. Chamberlain,3 Makoto Uchino.1 1 Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; 2Pharmacology, Sojo University, Kumamoto, Japan; 3Neurology, University of Washington School of Medicine, Seattle, WA. Duchenne muscular dystrophy (DMD) is an inherited severe muscle wasting disorder, and there is currently no effective treatment. DMD causes respiratory and/or cardiac failure and results in death at about 20 years of age. Lentiviral vectors are an efcient gene delivery tool for skeletal muscle bers as well as myogenic progenitor cells in vivo[/italic] and ex vivo[/italic]. The integration ability of lentiviral vectors is a huge advantage for targeting DMD myogenic cells, which require the missing gene product, dystrophin, to be expressed permanently, which can not be achieved with non-integrating vectors. We have shown that stable transduction of myogenic stem cells in Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy