152 Splice Site Strength and Nonsense Associated Exon Skipping in the DMD Gene Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S58 MUSCULO S[.]
MUSCULO-SKELETAL GENE & CELL THERAPY I 150 AAV Microgene Transfer Reveals the Cellular Motif for Dystrophin-Mediated Sarcolemmal Neuronal Nitric Oxide Synthase (nNOS) Localization Yi Lai,1 Yongping Yue,1 Dongsheng Duan.1 Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO Duchenne muscular dystrophy (DMD) is a severe inherited muscle disease caused by dystrophin deficiency Over the last few years, several highly abbreviated micro-dystrophin genes have been developed for DMD gene therapy Though promising, these microgenes remain suboptimal An important function of dystrophin is to anchor neuronal nitric oxide synthase (nNOS) to the sarcolemma We recently found that dystrophin spectrin-like repeats 16 and 17 (R16/17) are crucial for membrane-associated nNOS localization However, the underlying mechanism is not clear A better understanding of the nNOS recruiting motif in dystrophin may guide us develop more effective microgene therapy Each dystrophin spectrin-like repeat contains three α-helices Here we tested the hypothesis that R16/17-mediated nNOS anchoring depends on the correct α-helix phasing and composition To determine the impact of the α-helix phasing, we sequentially deleted one of the six α-helices of R16/17 in the ∆R2-15/∆R18-23/∆C microgene Adenoassociated virus (AAV) was used to introduce these microgenes to dystrophin-null mdx mice The original ∆R2-15/∆R18-23/∆C microgene efciently recruited nNOS to the membrane However, none of the modied microgenes restored sarcolemmal nNOS To determine the contribution of α-helix composition, we swapped each α-helix of R16/17 with the corresponding α-helix of repeat 18 and performed a yeast-two-hybrid assay Except for the rst α-helix of repeat 17, replacing other α-helices of R16/17 did not compromise nNOS interaction To conrm the in vitro nding, we engineered these chimeric repeats into the ∆R2-15/∆R18-23/∆C microgene and performed AAV gene transfer study In contrast to the yeast-two-hybrid result, in vivo screening suggest that the second, third α-helix of repeat 16 and the rst α-helix of repeat 17 are all required for anchoring nNOS In summary, our results suggest that the correct phasing and composition are critical for dystrophin-mediated sarcolemmal nNOS localization An intact R16/17 is essential for micro-dystrophin based DMD gene threrapy 151 Cell Therapeutic Approach to Duchenne Muscular Dystrophy Using Myogenic Differentiation of Multipotent Mesenchymal Stromal Cells Yuko N Kasahara,1 Hiromi H Kinoh,1 Hironori Okada,1 Jin-Hong Shin,1 Akiyo Nishiyama,1 Sachiko O Hosoyama,1 Michiko W Maeda,1 Akinori Nakamura,1 Takashi Okada,1 Shin’ichi Takeda.1 Department of Molecular Therapy, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan Background: Duchenne muscular dystrophy (DMD) is an incurable genetic disease with early mortality We studied gene and cell therapeutic approach to DMD in dogs using myogenic differentiation of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) to establish an efcient protocol Because of safety and utility, MSCs would make a larger contribution to clinical benet than iPS cells Since current methods of myogenic differentiation of MSCs are generally inefcient, we employed MyoD, the master switch protein for myogenic differentiation, to convert MSCs into myogenic cells We also investigated the strategies for cell expansion, and delivery route for effective cell transplantation in dystrophic dog Methods: Canine CD271-positive MSCs obtained from donor bone marrow cells were enriched by immunomagnetic isolation The dog leukocyte antigens (DLA) were analyzed to determine DLA- matched mating S58 pairs to achieve donor or recipient of allogeneic transplantation Cardiotoxin (CTX) was injected into tibialis anterior (TA) and extensor carpi ulnaris (ECU) muscles days before MSCs injection MSCs transduced with adenoviral vector expressing MyoD (AdMyoD) were injected into the CTX- injured TA (2x106 cells) and ECU muscles (1x106 cells) of recipient normal dog with oral administration of cyclosporine and mycophenolate mofetil We also injected or 2x106 of MSCs into DMD dog For intra-arterial administration, MyoD-transduced MSCs (5x106 cells) were administered into femoral artery with transient avascularization using a tourniquet The treated muscles were biopsied and analyzed histologically Results: CD271-enriched MSCs obtained from normal dog showed 20-fold higher growth expansion compared with CD271-depleted MSCs MyoD transduction of the CD271-enriched MSCs revealed in vitro myogenic differentiation and myotube formation MSCs transplanted to the DLA- matched recipient dog were observed as several clusters in the injected muscles weeks after the intramuscular injection The engraftment of MSCs was also successful with the immunosuppressant-free transplantation into DMD dog by the intramuscular injection Furthermore, extensive engraftment of MSCs was detected at the site of the CTX-injured muscle weeks after the intra-arterial injection Immunohistological analysis suggested that most of MSCs formed muscle-like tissues with the upregulation of developmental myosin heavy chain Conclusion: MyoD-transduced MSCs enabled more efcient realization of MSCs transplantation by the intramuscular as well as intra-arterial injection Although further study is required for effective differentiation in vivo, this strategy of MSCs propagation and treatment would be promising for the future DMD cell therapy 152 Splice Site Strength and NonsenseAssociated Exon Skipping in the DMD Gene Kevin M Flanigan,1 Diane M Dunn,3 Jerry R Mendell,1 Alan Pestronk,2 Julaine M Florence,2 United Dystrophinopathy Project Consortium, Robert B Weiss.3 Center for Gene Therapy, Nationwide Children’s Research Institute, Columbus, OH; 2Department of Neurology, Washington University, St Louis, MO; 3Department of Human Genetics, University of Utah, Salt Lake City, UT Understanding the molecular pathogenesis of DMD gene mutations is more compelling than ever considering evolving treatment strategies that include gene replacement therapy, exon skipping, and nonsense mutation readthrough Nonsense mutations can be associated with a spectrum of phenotypes: Duchenne Muscular Dystrophy (DMD) with loss of walking by age 12, Becker Muscular Dystrophy (BMD) with continued walking after age 15, and an intermediate (IMD) phenotype with loss of walking between ages 12 and 15 In some cases, a nonsense mutation has been shown to ablate an exon splice enhancer (ESE) or create an exon splice suppressor (ESS) motif, resulting in altered mRNA splicing with a resultant milder phenotype To assess the generality of this mechanism, we evaluated 166 unique nonsense mutations found in 210 patients with well-established phenotypes Among mutation sites in which mutation-induced exon skipping would be predicted to result in an out-of-frame transcript, only 5% resulted in BMD or IMD (n=4) and 95% resulted in DMD (n=77) In contrast, among mutations in an in-frame anking exon context, 18% (n=15) resulted in BMD or IMD, and 82% (n=70) resulted in DMD Thus, in BMD or IMD the exonic sequence context is typically in-frame (15/19) (P=.014 by Fisher’s exact test), although the presence of an in-frame exon context is not by itself predictive of BMD We calculated the splice site strengths of in- versus out-offrame exons using maximum entropy (MaxEnt), multiple dependence decomposition (MDD), rst order Markov model (MM), and a weight matrix model (WMM) On average, in-frame exons have weaker splice site (ss) signals than out-of-frame exons, most notably in the Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy MUSCULO-SKELETAL GENE & CELL THERAPY I 5’ss strength (MaxEnt p=0.006; MDD p=0.065; MM p=0.029; WMM p=0.049) The subset of in-frame exons that are enriched for BMD nonsense mutations have on average weaker 3’ss strength compared to the other in-frame exons (MaxEnt p=0.015; MDD p=0.015; MM p=0.053; WMM p=0.015) Thus, exons with in-frame BMD nonsense mutations appear to have the weakest splice site signals of all the DMD exons, as measured by the mean difference between consensus values for 3’ss and 5’ss strengths (MaxEnt: 13.21 vs 15.96 for inframe BMD vs in-frame DMD, and 13.21 vs 16.93 for in-frame BMD vs out-of-frame exons) In contrast, BMD mutations are not more frequently associated with either the ablation of ESE motifs or the creation of an ESS site than are DMD mutations (Mann-WhitneyWilcoxon rank sum test, p=0.7) Our data suggest that in BMD, ESE ablation or ESS creation signals are not the sole determinant of nonsense mutation-associated exon skipping, but rather that the effects of these exonic signal alterations are manifest on a background of weak intrinsic splice site signals within a restricted set of exons An improved understanding of DMD splice site metrics may shed light on the design of antisense oligonucleotides, and the interpretation of results from upcoming clinical trials of exon skipping 153 Long Term Antisense Treatment in Dystrophic Mouse Models for Duchenne Muscular Dystrophy Annemieke Aartsma-Rus,1 Christa L de Winter,1 Hans A Heemskerk,1 Judith C van Deutekom,2 Gert-Jan B van Ommen.1 Human Genetics, Leiden University Medical Center, Leiden, Netherlands; 2Proesensa Therapeutics, Leiden, Netherlands Antisense-mediated reading frame restoration is presently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD) In this approach, antisense oligoribonucleotides (AONs) induce specic exon skipping during pre-mRNA splicing of mutated dystrophin transcripts This is aimed to restore the disrupted open reading frame and allow synthesis of internally deleted, partly functional Becker-like dystrophin proteins The approach is theoretically applicable to over 70% of all patients, with exon 51 skipping being applicable to the largest group of patients (13% of all mutations) Proof of concept has been achieved in cultured muscle cells from patients carrying different mutation types, in the mdx mouse model, and recently in DMD patients in clinical trials after local or subcutaneous (systemic) treatment of PRO051, a 2’-O-methyl phosphorothioate modified AON In each case AON treatment resulted in skipping of the targeted exon and dystrophin restoration in the absence of adverse effects A subsequent trial where patients are treated systemically has recently been completed successfully and a 6-months followup trial using the most effective dosage is underway by Prosensa Due to AON turnover, repeated treatment is necessary Therefore, long term safety and efficacy of subcutaneous 2’O-methyl phosphorothioate AON treatment was tested in mouse models with varying levels of severity: mdx mice (mild phenotype) and mdx mice with one utrophin allele (mdx +/-; intermediate phenotype) Mice were treated with weekly SC injections of 200 mg/kg for up to months This was well tolerated during treatment and liver and kidney weights and serum parameters were similar for 4, 8, 12 and 24 week treated mice compared to saline treated controls at the end of treatment In both models treatment resulted in signicantly improved serum creatine kinase (marker for muscle quality) and rotarod running time (marker for muscle function) compared to the controls In the more severely affected mdx +/- mice the therapeutic effect was larger Time course experiments revealed that exon skipping levels increased for the rst 12 weeks, but remained constant after that Protein analysis revealed a similar pattern These results indicated that long term subcutaneous treatment with 2’-O-methyl phosphorothioate AONs is safe and efcient in Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy dystrophic mouse models, which is encouraging for future long term trials in patients, which are currently planned by Prosensa Therapeutics 154 Exon Exchange Approach To Repair Duchenne Dystrophin Transcripts Stéphanie Lorain, Cécile Peccate, Maëva Le Hir, Graziella Grifth, Thomas Voit, Luis Garcia Université Pierre et Marie Curie UMR S 974 – Inserm U974 – CNRS UMR 7215, Institut de Myologie, Paris, France Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences A number of trans-splicing molecules have already been proposed for replacing either the 5’ or the 3’ part of transcripts to be repaired Here, we show for the rst time the feasibility of RNA surgery by using a double trans-splicing approach allowing the specic substitution of a given mutated exon As a target, we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23) This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains We obtained accurate and efcient replacement of exon 23 in the mRNA target Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efciency up to 53% of repair These results demonstrate the possibility to x up mutated exons and more attractively refurbish deleted exons if required, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation Conversely to the well known exon skipping, ExChange has the advantage to be compatible with any type of mutations and more generally to a wide range of genetic conditions In particular, it allows addressing disorders caused by dominant mutations 155 Gentamicin Treatment of Duchenne Muscular Dystrophy Reinforces the Potential for Mutation Suppression Therapy Vinod Malik,1 Louise R Rodino-Klapac,1 Laurence Viollet,1 Cheryl Wall,1 Wendy King,1 Roula Al-Dahhak,1 Sarah Lewis,1 Christopher J Shilling,1 Janaiah Kota,1 John Hayes,2 John D Mahan,3 Katherine J Campbell,4 Brenda Banwell,5 Majed Dasouki,6 Victoria Watts,6 Kumaraswamy Sivakumar,7 Ricardo Bien-Willner,7 Kevin M Flanigan,1 Zarife Sahenk,1 Richard J Barohn,6 Christopher M Walker,4 Jerry R Mendell.1 Gene Therapy, The Research Institute at Nationwide Childrens Hospital, Columbus, OH; 2Pacic University, Forest Grove, OR; 3The Ohio State University, Columbus, OH; 4vaccines & Immunity, The Research Institute at Nationwide Childrens Hospital, Columbus; 5University of Toronto, Toronto, ON, Canada; University of Kansas, Kansas City, KS; 7Neuromuscular Research Center, Scottsdale, AZ Duchenne Muscular Dystrophy (DMD) is a severe X-linked genetic muscle disease caused by nonsense mutations of the dystrophin gene in about 15% of cases Pre-clinical studies in mdx mice demonstrate that gentamicin suppression of premature termination codons allows readthrough, thereby translating the full length protein Clinical attempts to reproduce these ndings in DMD patients have been inconsistent In the current clinical study we have attempted to address several issues: 1) the biopotency of gentamicin comparing stop codon DMD subjects with patients harboring frameshift mutations; 2) the feasibility of long-term gentamicin administration considering that readthrough would be an ongoing requirement; 3) the percent dystrophin expression required to provide clinically S59 ... of the mdx dystrophin gene enclosing the mutated exon (exon 23) This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains We obtained... weak intrinsic splice site signals within a restricted set of exons An improved understanding of DMD splice site metrics may shed light on the design of antisense oligonucleotides, and the interpretation... premature termination codons allows readthrough, thereby translating the full length protein Clinical attempts to reproduce these ndings in DMD patients have been inconsistent In the current clinical