131 In Utero Delivery of Adeno Associated Virus Vector Encoding Human Mini Dystrophin Leads to Functional Correction in Dystrophin Deficient Mice (PMO) chemistryvia fluorescencemicroscopy, RT PCRlnest[.]
(PMO) chemistryvia fluorescencemicroscopy, RT-PCRlnestcd-PCR analysis, as well as flowcytommetry.Aller initialselection of AONs, the effect ofoverall AON length was also determined on the culture system The efficient AONs selected were further tested with normal human myoblasts and relevant patient fibroblasts (with hEx5l deletion; further skipping of E50 can restore the reading frame) The results showed thatAONs selected with the reporter system are highly effective for specific exon skipping in all cells, Our results demonstrate that the in vitro cell culture-based system is a valuable tool for the screening of effective AONs with high specificity and reliability, both of which are essential for clinical trials involving antisense therapy in DMD 129 A Myoblast Expansion System for Improving the Efficiency of Autologous Stem Cell Therapy To Treat Muscular Dystrophy Sheng Li,' Brent Fall,' Miki Haraguchi,' Jeffrey S Chamberlain I 'Senator Paul D Wellstone Muscular Dystrophy Co-operative Research Center, Department ofNeurology; The University 0/ Washington School ofMedicine, Seattle, IE,/ Autologous stem cell-based transplantation is a promising approach to treating inherited muscle disorders A successful strategy would be facilitated by regimes that allow tor expansion both in vitro and in vivo of cells with myogenic potential A previous study demonstrated that a chemical inducible dimerizer (CID), AP20 187, could maintain in vitro proliferation of myoblasts expressing P36VPGFR-I, a chimeric protein composed of the cytoplasmic phosphorylation domain of fibroblast growth factor receptor (FGFR-I) and a mutated dimerization domain of FK506 binding protein (F36V) Here, we generated a lentiviral vector carrying a bicistronic expression cassette composed ofa microdystrophin/GPP fusion gene and the F36VFGFR-1 gene under the control of a synthetic muscle-specificpromoter.The mdx rnyoblaststransduced with this vector were prevented from differentiating and were expanded in culture medium containing AP20187, but lacking FGF-2 When intramuscularly transplanted into mdx tibialis anterior muscles, these AP20 187-expanded cells formed large clusters of myofibcrs expressing microdystrophin/GFP and the developmental isoform of myosin heavy chain.Although CID-cxpanded myoblasts diffcrcntiated to form rnyotubes and myofibers in skeletal muscle, we found that in culture, residual AP20187 retained in those CID-expanded cells dramatically delayed myotube formation These observations imply that exogenous control of FGFR-I activation in myoblasts can be used to prevent terminal differentiation and enable largescale expansion of myogenic precursors, potentially increasing the efficiency of myoblast engraftment in muscle disorders 130 Independent Canine Models of Duchenne Muscular Dystrophy Due to Intronic Insertions of Repetitive DNA Bruce F Smith,' Joe N Kornegay- Dongsheng Duan ' 'Scott-Ritchey Research Center; Auburn University, Auburn, AL; lSchool 0/Medicine, University ofNorth Carolina - Chapel Hill, Chapel Hill, NC; 3School ofMedicine, University ofMissouri, Columbia , Ala Duchenne muscular dystrophy (DMD) is the most common Xlinked disease and inherited myopathy of humans As such, devclopment of effective gene therapy for DMD has been and continues to be a high priority A number of animal models have allowed the testing of novel approaches in order to determine their validity prior to application to human patients Of these, the canine model most closely recapitulates the clinical presentation, immune system responses and body mass of human patients Several canine mutations have been identified, althoughthe Golden Retrieverremainsthe Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © The American Soci ety o r Gene Therapy best characterized and therefore most commonly employed model Additional canine models would further improve the utility of the dog as a model system as these models would mimic the variety of challenges seen with human patients, including variable transcription, the presence or absence of epitopes and the effect of residual mutant or "revertant" protein We have characterized two additional canine models of DMD at the level of histology, morphology and molecular basis These models were identified in the Labrador Retriever and Welsh Corgi breeds In both cases, affected animals can be identifiedat birth by elevated creatine kinase levels Both Welsh Corgis and Labrador Retrievers have a relentlessly progressive and ultimately fatal course of disease Both models are dystrophin deficient except for rare revertant fibers They display prominent skeletal muscle pathology identical to these found in human patients such as variable fiber size, central nucleation, fiber splitting, fatty infiltration, macrophage infiltration, fibrosis and caleification We have identified the molecular basis of each model as the inclusion of repetitive sequence elements creating novel exons in the cDNA In both cases these elements have been inserted into introns (intron 13 for the Welsh Corgi and intron 19 for the Labrador Retriever), activating splice acceptor sites already present in the normal intron sequence In bothcases, the inserted sequences contain in-framestop codons leading to early termination of translation The characterization ofthe mutations and morphologic features of each ofthese new models provides the information required to employ these models in gene therapy studies These models provide important alternatives to the available models and they will broaden our understanding of how relevant approaches will function in the face ofdifferent mutations and clinical manifestations in the human population 131 In Utero Delivery of Adeno-Associated Virus Vector-Encoding Human Mini-Dystrophin Leads to Functional Correction in Dystrophin Deficient Mice Anthony Y Tsai,' Sasha Bogdanovich,' Christina F Hughes,' Masayuki Endo, I Jesse Vrecenak,I Jeremy Traas,' Philip Zoltick, I Tejvir S Khurana.' Tim Brazelton, I Alan W Flake.' 'Surgery; Children's Hospital ofPhiladelphia, Philadelphia, PA; 'Physiology; University 0/Pennsylvania, Philadelphia, PA Introduction: Duchenne muscular dystrophy (DMD) is the most common disabling and lethal congenital muscle disorder Patients with DMD experience progressive muscle degeneration and weakness until they succumb to respiratory or cardiac failure Currently there is no cure for DMD Human mini-dystrophin with truncation of the repeating rod domain has been shown to amelioratedystrophic histopathology and restores membrane integrity.Adeno-associated virus serotype (AAV2/9)has also been shown to have high muscle specificity Here, we investigated the possibility of using in utero delivery of AAV2/9 vector-mediated mini-dystophin to achieve long-term functional correction of'dystrophin deficient mdx mice Methods: Fetuses from pregnant mdx mice underwent vitelline vein injectionat E 14.5 withAAV2/9.CMV.mini-dystrophin Specific skeletal muscles, cardiac muscle, and brain and liver tissue from treated and age-matched mdx control mice were harvested on week 5, 10 and 20 Human mini-dystrophin expression was detected by immunohistochemistry (IHC) of frozen sections and quantified by real-time PCR (Q-PCR) Extensor digitorum longus (EDL) muscles of the treated and control mice were used in physiological studies to measure force of maximal twitch, tetanus and eccentric force drop between first and fifth contractions (ECC) Results: Expression of human mini-dystrophin was detected in week 5, 10, and 20 samples using IHC with human specific antibody with no significant difference in the proportion of transduction among the time points Q-PCR of heart, TA and soleus at the 20-week time point using the human dystrophin probe (hDys) with the ribosomal ISS S51 probe as the endogenous control detected a normalized hDys/l8S RNA ratio of 4.46±3.04, 2.29±1.12, and 2.76±3.86, respectively Functional studies showed that ECCs demonstrated significantly (P