74 Addition of Peptide Therapy To Inhibit NF κB Activation to AAV Serotype 9 Mini Dystrophin Gene Transfer To Treat Muscular Dystrophy in mdx Mice Molecular Therapy Volume 19, Supplement 1, May 2011[.]
MUSCULO-SKELETAL GENE & CELL THERAPY and RTC14, were recently identified by a luciferase-independent high-throughput screening assay and were shown to have potential therapeutic functions in the treatment of nonsense mutations We have tested the ability of RTC13 and RTC14 to restore dystrophin expression into skeletal muscles of the mdx mouse model for Duchenne muscular dystrophy (DMD) Intramuscular injections of RTC13, promoted read-through of the mdx UAA stop codon more efficiently than gentamicin, PTC124 or RTC14 making it our lead drug candidate When administered systemically, RTC13 was shown to restore dystrophin protein in different muscle groups, including diaphragm and heart Improved muscle strength was detected in all treated animals and was accompanied by a significant decrease in creatine kinase (CK) levels demonstrating that the compound was able to slow down muscle degeneration and turnover No signs of toxicity were detected in mdx after prolonged administration of RTC13 demonstrating that the compound was well tolerated in mice The levels of direct bilirubin (DBIL), blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were significantly decreased in RTC13-treated mice as compared to untreated mdx or mdx mice that received vehicle alone confirming that restoration of dystrophin expression in muscles was able to ameliorate some of the secondary pathology associated with the disease in mdx Structure activity relationship (SAR) studies were used to optimize the molecular structure of RTC13 and to identify a derivative that meets optimal safety profiles while still maintaining maximal read-through activity These results advance the development of RTC13 as an effective drug candidate for DMD They also offer hope for the treatment of numerous other genetic disorders due to nonsense mutations and premature termination of protein synthesis 72 Effective Limb Transduction and Phenotypic Correction after Injection of rAAV8-U7 snRNA in GRMD Dogs Caroline Le Guiner,1,2 Marie Montus,1 Laurent Servais,3 Luis Garcia,3 Yves Fromes,1,3 Jean-Yves Hogrel,3 Pierre Carlier,3 Yan Cherel,4 Philippe Moullier,1,2 Thomas Voit,3 The AFM-Sponsored Duchenne Consortium.1,2,3,4 Genethon, Evry, France; 2INSERM UMR 649, Nantes, France; Institut de Myologie, Paris, France; 4INRA UMR 703, Nantes, France In Duchenne Muscular Dystrophy (DMD) the selective removal by exon skipping of exons flanking an out-of frame mutation in the dystrophin messenger can result in in-frame mRNA transcripts that are translated into shorter but functionally active dystrophin The goal of our project was to determine in GRMD, the effective dose of our therapeutic product defined as a recombinant Adeno-Associated Virus serotype (rAAV8) expressing a modified U7 snRNA specific for the skipping of exons to 10 of the GRMD dystrophin transcript The mode of delivery was the locoregional high-pressure intravenous (IV) injection of a forelimb Several groups of GRMD dogs were exposed to different rAAV8-U7snRNA doses Each dog was followed ∼3 months after injection The primary outcomes were the restoration of dystrophin expression and the improvement of the tissue pathology in the injected limb compared to the controlateral limb The secondary outcomes were the muscle strength correction, the biodistribution and shedding patterns as well as the immune response against rAAV8 capsid and dystrophin Our preliminary results suggest a dose effect of our therapeutic rAAV Injection of 2,5E13vg/kg and of 5E12vg/kg of our vector was able to restore 50 to 80% of Dystrophin expression in the injected limb This expression of a semi-functional dystrophin resulted in improvement of tissue morphology as well as of several functional and MRI parameters No tissue inflammation occurred following the procedure We built a unique network of laboratories with complementary skills to deliver a Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy GLP-compliant set of preclinical data to further define the regulatory toxicology studies The organization of our network and the results obtained in our GRMD dogs study will be presented This project is supported by AFM (Association Franỗaise contre les Myopathies) and by ADNA (Advanced Diagnostics for New Therapeutic Approaches), a program dedicated to personalized medicine, coordinated by Institut Mérieux and supported by research and innovation aid from the French public agency, OSEO 73 Inhibition of CD26 Activity Enhances Engraftment of Donor Cells to Regenerating and Dystrophic Skeletal Muscle Maura H Parker,1 Carol Loretz,1 Rainer Storb,1,3 Stephen J Tapscott.2,4 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA; 3Department of Medicine, University of Washington, Seattle, WA; 4Department of Neurology, University of Washington, Seattle, WA Muscle-derived cell transplantation has the potential to effectively treat many human diseases, including muscular dystrophy A variety of cell populations engraft into skeletal muscle of mdx mice, effectively restore dystrophin expression and reconstitute the satellite cell pool Yet, a direct and quantitative comparison of engraftment to determine the most effective cell population is lacking We have developed a canine-to-mouse xenotransplantation model to rapidly and quantitatively compare canine muscle cell engraftment Specifically, we demonstrate that canine muscle derived cells engraft into regenerating mouse muscle, and engraftment is quantifiable and consistent The canine-to-mouse model allows us to quantitatively compare cell populations and modulating factors, and establish priority for transplantation experiments using a clinically relevant immune tolerant cxmd canine model of muscular dystrophy We used the xenotransplant model to show that canine muscle derived cells sorted for expression of CXCR4 not display a greater level of engraftment when compared to a mixed cell population However, pretreating a mixed cell population with diprotin A, a positive modulator of CXCR4-SDF-1 binding, significantly enhances engraftment of donor cells to the mouse satellite cell niche Translating these results to the immune tolerant canine, we demonstrate that injection of diprotin treated donor cells results in a significantly increased number of muscle fibers expressing dystrophin as comapred to untreated cells Temporal regulation of CXCR4/SDF-1 binding may be an important means of expanding the effective range of engraftment after transplantation 74 Addition of Peptide Therapy To Inhibit NFκB Activation to AAV Serotype Mini-Dystrophin Gene Transfer To Treat Muscular Dystrophy in mdx Mice Daniel P Reay,1 Gabriela A Niizawa,1 Jon F Watchko,2 Molly Daood,2 Eugene Raggi,1 Paula R Clemens.1,3 Neurology, University of Pittsburgh, Pittsburgh, PA; 2Pediatrics, Magee-Women’s Research Institute, Pittsburgh, PA; 3Neurology Service, Department of Veteran’s Affairs Medical Center, Pittsburgh, PA Systemic gene transfer using serotype adeno-associated vectors (AAV9) is promising for treatment of preclinical models of Duchenne muscular dystrophy (DMD) The ability to achieve systemic vector delivery circumvents a significant hurdle presented by the widespread distribution of skeletal muscle that is best accessed through the circulation However, a limitation of systemic gene vector delivery is that gene transduction levels vary among muscle groups The addition of complementary therapy could provide 1) a treatment effect S29 MUSCULO-SKELETAL GENE & CELL THERAPY independent of gene transfer and 2) facilitation of successful gene transfer Pathological activation of the nuclear factor κB (NF-κB) signaling pathway has emerged as an important cause of dystrophic muscle changes in muscular dystrophy Furthermore, activation of NF-κB may also inhibit gene transfer by promoting inflammation in response to the transgene or vector Therefore, we hypothesized that the addition of NF-κB inhibition in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD The NF-κB Essential Modulator (NEMO) binding domain (NBD) peptide, which is utilized in this study to inhibit NFκB, is synthesized as a fusion peptide with a protein transduction domain of lysines (8K) to facilitate intra-cellular delivery An AAV9 vector carrying a human codon-optimized mini-dystrophin gene under control of the cytomegalovirus promoter was given as a single intraperitoneal injection (1x1011 gc/pup) at age 2-3 days to 16 mdx mice (experimental mice) Starting at age weeks, of the experimental mice were given intraperitoneal injections of 8K-NBD peptide dosed at 10mg/kg, times per week for weeks The second group of experimental mice received sham intraperitoneal saline injections Control groups (n=8/group) included normal C57BL10 and disease control untreated mdx mice At sacrifice, ex vivo physiological studies of diaphragm force production were performed prior to histological and biochemical assays of diaphragm and limb muscle tissue In mdx mice treated with the combination of 8K-NBD peptide and AAV9 mini-dystrophin gene delivery, the quadriceps muscle demonstrated increased levels of recombinant dystrophin expression (84% of fibers expressing, as compared to 56% with AAV9 minidystrophin gene delivery alone) suggesting that 8K-NBD treatment promoted an environment in muscle tissue conducive to higher levels of recombinant dystrophin expression Indices of necrosis and regeneration were diminished with either AAV9 gene delivery alone or in combination with 8K-NBD treatment In diaphragm muscle, transgene expression was sufficiently high (>95% of muscle fibers expressed dystrophin) that marked improvements in histological and physiological indices were comparable in the treatment groups The data support benefit from 8K-NBD treatment to complement gene transfer therapy for DMD in muscle tissue that receives incomplete levels of transduction by gene transfer 75 RNA Interference Improves Myopathic Phenotypes in Mice Over-Expressing Facioscapulohumeral Muscular Dystrophy Region Gene (FRG1) Lindsay M Wallace,1,2 Sara E Garwick-Coppens,1 Scott Q Harper.1,2,3 Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; 2Molecular, Cellular and Developmental Graduate Program, The Ohio State University, Columbus, OH; 3Pediatrics, The Ohio State University, Columbus, OH Muscular dystrophies, and other diseases of muscle, arise from recessive and dominant gene mutations Gene replacement strategies may be beneficial for the former, while gene silencing approaches may provide treatment for the latter In the last two decades, muscle-directed gene therapies were primarily focused on treating recessive disorders This disparity at least partly arose because feasible mechanisms to silence dominant disease genes lagged behind gene replacement strategies With the discovery of RNA interference (RNAi) and its subsequent development as a promising new gene silencing tool, the landscape has changed In this study, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo We tested the potential of AAV-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) Region Gene (FRG1), to correct myopathic features in mice expressing toxic levels S30 of human FRG1 (FRG1-high mice) We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1-high mice Specifically, muscles transduced with FRG1-targeted microRNAs (miFRG1) were normal in size, showed no fibrosis or fat deposition, and had no evidence of myofiber degeneration or regeneration Moreover, grip strength testing showed that miFRG1-treated animals were as strong as wild-type mice, while control-treated or untreated animals remained significantly weaker Our results support the feasibility of using RNAi to target other FSHD candidate genes, including DUX4, which has recently emerged as the leading pathogenic insult underlying FSHD Moreover, this approach potentially applies to as many as 37 different gene mutations responsible for myopathies inherited as autosomal dominant disorders 76 Sustained Expression of Canine MicroDystrophin and Amelioration of Muscle Function in Dystrophic Dogs Following Large Scale AAVMediated Treatment Zejing Wang,1,2 Rainer Storb,1,2 Donghoon Lee,3 Christine Halbert,4 Martine K Childers,5 Glen Banks,6 James Allen,6 Eric Finn,6 Martin Kushmeric,3 Dusty Miller,4 Jeffery S Chamberlain,6 Stephen J Tapscott.1,4,6 Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Medicine, University of Washington, Seattle, WA; 3Radiology, University of Washington, Seattle, WA; 4Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Neurology and Regenerative Medicine, Wake Forest University, Charlotte, NC; 6Neurology, University of Washington, Seattle, WA Adeno-associated viral (AAV) vectors as gene delivery vehicles have shown promise both in preclinical studies and clinical trials for a number of acquired and inherited diseases, including Duchenne Muscular Dystrophy (DMD) We and others have shown that dogs and humans mount immune responses against all tested AAV serotypes, which compromised vector delivery and prevented sustained therapeutical gene expression We have developed a transient immunosuppressive regiment consisting of anti-thymocyte globulin (ATG), cyclosporine (CSP), and mycophenolate mofetil (MMF), which was effective in preventing immune responses to AAV and enhanced AAV6-mediated gene delivery to canine skeletal muscle Here, we demonstrate that long term expression for years of a species – specific, functional canine-micro-dys can be achieved in large scale in skeletal muscles of DMD dogs with ATG/CSP/MMF Moreover, for the first time we achieved amelioration of muscle histology and function as a result of sustained expression of the therapeutic gene as demonstrated both by non invasive magnetic resonance imaging (MRI) and kinametic gait analysis In order to identify an even more effective and less toxic immunosuppressive regimen, we have begun studies using molecules that block T-cell costimulation Further, we are also examining AAV vectors made by a modified production method that minimizes aberrant packaging of the capsid gene to further reduce immunogenicity in dogs In conclusion, the finding of long term dystrophin expression in DMD dogs given a short course of standard immunosuppression opens the possibility of translating these strategies to a human DMD trial Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... the addition of NF- κB inhibition in muscle would complement the therapeutic benefits of dystrophin gene transfer in the mdx mouse model of DMD The NF- κB Essential Modulator (NEMO) binding domain... important cause of dystrophic muscle changes in muscular dystrophy Furthermore, activation of NF- κB may also inhibit gene transfer by promoting inflammation in response to the transgene or vector Therefore,... performed prior to histological and biochemical assays of diaphragm and limb muscle tissue In mdx mice treated with the combination of 8K-NBD peptide and AAV9 mini- dystrophin gene delivery, the