772 Novel Functional Optical Imaging of Bone Repair in a Calvaria Defect Model Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S294 MUSCULO[.]
MUSCULO-SKELETAL GENE & CELL THERAPY II outcomes Bohannon RW Reference values for the Timed Up and Go Test: A Descriptive Meta-Analysis Journal of Geriatric Physical Therapy, 2006;29(2):64-8 769 AAV-Based shRNA Silencing of NF-κB Ameliorates Muscle Pathologies in mdx Mice Qing Yang,1,2 Ying Tang,1 Kara Imbrogno,1 Aiping Lu,1 Anmin Chen,2 Fengjin Guo,2 Freddie H Fu,1 Johnny Huard,1 Bing Wang.1 Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA; Orthopaedic Surgery, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, Hubei, China Duchenne muscular dystrophy (DMD) is an X-linked genetic muscle disease affecting of every 3500 male births Chronic inflammation, promoted by an up-regulated NF-kappaB (NF-κB) pathway, plays a key role in Duchenne muscular dystrophy (DMD) patients’ pathogenesis Higher immune alertness, due to chronic inflammation and immune effector cell infiltration, challenges recombination adeno-associated viral (AAV) vector-mediated gene replacement treatment for DMD, especially in large animal models and clinical trials We and others found that blocking the NF-κB pathway could be a viable solution to promote muscle regeneration and to decrease necrosis in mdx mice (a murine DMD model), by using transgenic and AAV-mediated transfer (dominant-negative forms of IKKα & β) approaches, without any side effect Based on the small interference RNA (siRNA) technology used in this study, we examined whether an AAV vector carrying small hairpin RNA (shRNA) targeting the NF-κB/p65 subunit, a major subunit of NF-κB associated with inflammation in mdx mice, inhibits NF-κB activation as well as ameliorates pathological necrosis and regeneration in the locally-treated gastrocnemius (GAS) muscle of and month old mdx mice At month post-treatment, NF-κB/p65 levels in locally-treated muscles were significantly decreased by the efficient transfer of specific shRNA in both of the different treated age groups This led to remarkable decreases in necrosis and myofiber damage compared to mdx mice treated by a phosphate-buffered saline (PBS) or a control AAV vector containing a scrambled shRNA The shRNA silencing of the NF-κB pathway diminished the pathological myofiber regeneration in young mdx mice treated at month of age, but it enhanced the capacity of myofiber regeneration in old mdx mice treated at months of age Moreover, quantitative analysis revealed that central nucleation of the myofibers of treated young mdx muscles was reduced from 67% to 34%, but centrally-located nuclei were not significantly reversed to the peripheral position in treated old mdx mice Our findings indicate that AAV-mediated NF-κB/p65-shRNA has the capacity to ameliorate muscle pathologies in mdx mice by degrading mRNA of the NF-κB/p65 subunit 770 Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse Tomohiro Suga,1 En Kimura,1 Yuka Morioka,2 Masahito Ikawa,2 Sheng Li,3 Katsuhisa Uchino,1 Yuji Uchida,4 Satoshi Yamashita,1 Yasushi Maeda,1 Jeffrey S Chamberlain,3 Makoto Uchino.1 Neurology, Kumamoto University Graduate School of Medical Sciences, Kumamoto, Japan; 2Research Institute for Microbial Diseases, Osaka University, Suita, Japan; 3Department of Neurology, University of Washington School of Medicine, Seattle; 4Pharmacology, Division of Life Science, Faculty of Pharmaceutical Sciences, Sojo University, Kumamoto, Japan Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed, but poorly analyzed The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, however it has not been studied in striate muscle We injected a lentiviral vector carrying an MSCVS294 EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles We also generated lentiviral-mediated transgenic mice carrying the MSCVEGFP cassette and detected transgene expression in striated muscles LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in type I and IIA, intermediate in type IID/X, and lowest in type IIB fibers The soleus and diaphragm muscles, consisting mainly of type I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice The Lvmediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes 771 Noninvasive Monitoring of Whole Body Muscle Function in Dystrophin-Deficient Dogs Jin-Hong Shin,1 Brian B Greer,1 Chady H Hakim,1 Zhihai He,2 Dongsheng Duan.1 Molecular Microbiology & Immunology, University of Missouri, Columbia, MO; 2Electrical and Computer Engineering, University of Missouri, Columbia, MO Dystrophin-deficient dogs are highly valuable tools for evaluating gene/cell therapies to treat Duchenne muscular dystrophy (DMD) Unfortunately, robust muscle functional end points are poorly defined in the canine DMD models Here, we described a set of non-invasive methods for whole body muscle function analysis in normal and affected dogs Briefly, the gait and voluntary activity were recorded using high-resolution digital video recorders Gait recoding was repeated three times on three different days for each dog Voluntary activity monitoring was performed under low-lux red lighting between 7pm and 5am for each dog The stride length, stride speed, range of the motion and overall activity were analyzed using a customer-developed program Three normal and four affected dogs of a litter were examined Affected dogs showed significantly reduced stride length (affected, 35.7 ± 8.5 cm; normal, 80.2 ± 6.1 cm; p