128 screening of antisense oligonucleotides for human dystrophin exon skipping in a GFP reporter culture system with high sensitivity and specificity

128 Screening of Antisense Oligonucleotides for Human Dystrophin Exon Skipping in a GFP Reporter Culture System with High Sensitivity and Specificity 125 Measurement of Flow Mediated and Nitric Oxide[.]

125 Measurement of Flow-Mediated and Nitric Oxide Synthase-Dependent Vasodilation in Living Rats Using High Resolution Ultrasound mass Alternatively, Dp 116 may contribute to the structural integrity of myofibers via non-canonical binding of Dp 116 to the actin cytoskeleton Christian Heiss , Richard E Sievers, Nicolas Amabile, Shobha Natarajan, Yerem Yeghiazarians, Matthew L Springer 1Department ofMedicine, Division ofCardiology, University of California, San Francisco, San Francisco, CA 127 Functional Capacity of Dystrophins NTerminal Actin Binding Sequences In humans, endothelial function serves as a surrogate marker for cardiovascular health and is measured as changes in arterial diameter after temporary ischemia (flow-mediated dilation; FMD) However, in rodent models of vascular disease, direct measurements of vascular function have been limited to studying isolated blood vessel fragments ex vivo Here, we present an FMD-related approach to study conduit artery vasodilation in anesthetized rats Diameter and Doppler-flow measurements were obtained from the femoral artery using high -resolution ultrasound and automated analysis software We observed dose-dependent vasodilation using both endotheliumdependent and endothelium-independent pharmacologic vasodilators Transient surgically-induced hindlimb ischemia led to reactive hyperemia with sequential flow velocity increase and femoral artery dilation, the latter of which was completely abolished by NO-synthase inhibition Reactive hyperemia was mimicked by adenosineinduced flow increase Repeated measurements were reproducible on the same day and at one month To demonstrate the applicability ofthis approach in a model ofendothelial dysfunction, we show that FMD is significantly reduced in older animals To our knowledge, this is the first integrative physiologic model to reproducibly study FMD in living rodents Notably, this approach demonstrates that the physiology of rodent vascular reactivity is concordant with that of humans, validating its use for the study of gene-based and other therapeutic modulators of vascular function GENE THERAPY FOR MUSCULAR DYSTROPHIES 126 Dp116 Expression in Dystrophin/Utrophin Double Knockout {mdxiutm -) Mice Restores Muscle Mass and Viability but Does Not Correct Dystrophic Pathology Andrea L H Arnett,' Luke M Judge,' Jeffrey S Chamberlain.' 'Molecular and Cellular Biology, Medical Scientist Training Program, University ofWashington, Seattle; 2Neurology, University ofWashington, Seattle Mice deficient in both dystrophin and utrophin (mdx.utrnr) exhibit a phenotype similar to that seen in DMD patients, including severe muscle wasting, skeletal deformities, joint contractures, and premature death In these mice, the absence of both dystrophin and utrophin prevents assembly ofthe dystrophin-glycoprotein complex (DGC) We previously generated transgenic mice with skeletal muscle expression of Dpl16 and have introduced this transgene onto the md:r utrn:': background Dp116 is a non-muscle isoform of dystrophin that lacks the actin-binding domain and the majority of the rod domain found in the full length dystrophin isoform Thus, it is postulated that Dp 116 has no mechanical link to the actin cytoskeleton, but can still assemble and stabil ize the DGC at the sarcolemma We now report that Dp 116 expression can dramatically increase both muscle mass and lifespan in mdxtutrn" and can delay both formation and progression of kyphosis and joint contractures However, histological examination of muscle from these transgenic mice reveals signs of muscular dystrophy similar to that seen in muscles of dystrophin-deficient (mdx) mice, Our results provide compelling evidence that dystrophin and the DGC participate in signaling mechanisms that are critical for maintenance of muscle S50 Glen B Banks, I Paul Gregorevic, I James M AlIen,1.2 Eric E Finn,' Jeffrey S Charnberlain.l-P 1Department ofNeurology, University ofWashington , Seattle, 11'11; 2Department ofMedicine, University ofWashington, Seattle, WA; 'Department of Biochemistry: University ofWashington Seattle, Wit Duchcnne and Becker muscular dystrophies are caused by mutations in the dystrophin gene Mutations in the N-terminal actin-binding domain (ABO I) of dystrophin typically decrease dystrophin expression and cause clinically more severe phenotypes than similar mutations elsewhere in the protein Because of the large reduction in protein expression, thc functional capacity of actin binding sequences in the N-terminal domain is not clear ABO I contains three actin-binding sequences (ABS 1-3) In thc present study we test the pathophysiological effects of in-frame actin-binding sequence deletions from microdystrophin (L'1R4R23/L'1CT) when expressed in muscles ofdystrophin-deficient mdx mice We delivered microdystrophin into the tibialis anterior muscles of 2-day-old mdx mice using recombinant adcno-associatcd viral vectors pscudotypcd with the serotype-6 capsids, Mierodystrophin prevented muscle degeneration and loss ofspecific force generating capacity, and partially protected muscles from contraction-induced injury when evaluated at months of age Expression ofmicrodystrophin lacking ABS3 (ABS I ,2~Dys) or ABS2 and (ABS I ~Dys) in the N-terminal domain did not prevent loss of specific force nor protect muscles from contraction-induced injury Furthermore, expression of dystrophins with these N-terminal deletions reduced the overall pereentagc of muscle fibers that were protected from degeneration, compared with the non-deleted microdystrophin Therefore, dystrophin-based structs appear to require an intact ABO I to restore the contractile properties of skeletal muscle and prevent ongoing muscle degeneration 128 Screening of Antisense Oligonucleotides for Human Dystrophin Exon Skipping in a GFP Reporter Culture System with High Sensitivity and Specificity Ehsan Benrashid,' Saafan Malik,' Allen Zillmer,' Randy J Thresherf Jeffrey Rosenfeld,' Qi Long Lu IMcCol/-Locf wood Laboratoryfor Muscular Dystrophy Research, Carolinas Medical Center; Charlotte NC; 2Animal Models Core Facility, University ofNorth Carolina Chapel Hill NG Frameshift mutations in the dystrophin gene, located on the X chromosome (Xp21), can result in the progressive muscle wasting disease Duchenne's Muscular Dystrophy (DMD), which affects-a in 3500 live male births Antisense oligonucleotide (AON) therapy has previously been shown to selectively induce the skipping of particular exons both in vitro and in vivo, by targeting exon splicing enhancer (ESE) regions, 3'-, and 5' -splice sites The DMD phenotype can thus be alleviated to the milder Becker's Muscular Dystrophy (BMD) form Due to limitations in the quantification of AON efficacy via RT-PCR and nested PCR assay systems, as well as practical limitations of primary cultures for AON screening, this study employed a GFP reporter culture system to provide for efficient and high throughput screening ofnumerousAON compounds for human dystrophin exon 50 skipping, which could rescue approximately 8% ofDMD mutations AON screening was performed using both 2' -O-mcthyl phosphorothioate (20Mc) and morpholino Molecular Therapy Volume 15.Supplement t••\by 2007 C op~Ti~ht © The American $