71 Restoration of Dystrophin Expression in the mdx Mouse Model for Duchenne Muscular Dystrophy (DMD) Induced by RTC13 Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Socie[.]
MUSCULO-SKELETAL GENE & CELL THERAPY Musculo-skeletal Gene & Cell Therapy 69 Exploring the Potential of Plasma Pheresis To Circumvent Pre-Existing Antibodies to AAV8 on Transgene Expression Following Targeted Vascular Delivery Louis G Chicoine,1,2 Louise R Rodino-Klapac,1,2 K Reed Clark,1,2 Chrystal L Montgomery,1,2 Thomas J Preston,1 William G Bremer,1,3 Katherine J Campbell,1,3 Zarife Sahenk,1,2,4 Paul T Martin,1,2 Christopher M Walker,1,3 Jerry R Mendell.1,2,4 Pediatrics, The Ohio State University/Nationwide Children’s Hospital, Columbus, OH; 2Center for Gene Therapy, Research Institute at Nationwide Children’s Hospital, Columbus, OH; Center for Vaccines and Immunity, Research Institute at Nationwide Children’s Hospital, Columbus, OH; 4Neurology, The Ohio State University/Nationwide Children’s Hospital, Columbus, OH Gene therapy for muscle disease has taken several positive steps forward We recently reported sustained AAV mediated alphasarcoglycan (SGCA) gene expression for as long as months in of limb girdle muscular dystrophy (LGMD2D) treated patients (Mendell et al 2010) The one patient that did not express SGCA was found to have high AAV neutralizing antibodies prior to gene transfer With vascular gene therapy to treat multiple muscles as the next logical step clinically, impediments may lie with the immune response and the presence of pre-existing antibodies to various AAV serotypes We tested the hypothesis that pre-existing antibodies to AAV8 would attenuate transgene expression following vascular delivery of two potentially therapeutic transgenes AAV8.MCK micro-dystrophin.Flag and AAV8.MCK.Galgt2 in a large study of 72 non-human primates Prior to gene transfer, all animals were pre-screened for binding antibodies to AAV8 Those animals with a titer of 1:100) were considered positive Animals were stratified into positive and negative groups with scheduled sacrifice times at and months (n=9 per group) Vascular delivery to the gastrocnemius muscle of an isolated limb (Rodino-Klapac et al 2010) was performed on each animal where x 1012 vg/kg of the appropriate vector was given None of the animals suffered noticeable edema or adverse effects from the procedure Three or six months after transfer, the macaques were euthanized, and the gastrocnemius muscle was harvested Samples of the proximal, central and distal muscle were stained with antiFLAG or CT2 antibody The contralateral gastrocnemius served as a negative control Gene expression was visualized in all subjects and subjects without pre-existing AAV8 antibodies demonstrated significantly higher transgene expression than subjects with preexisting antibodies (62% vs 33% muscle fibers transduced, P ≤ 0.001) Regression analysis confirmed a direct inverse correlation between the AAV8 antibody titer and the percent gene expression From these data we conclude that the presence of pre-existing antibodies to the vector will attenuate transgene expression Plasma pheresis studies appeared to optimize transgene expression and this procedure would potentially be used to increase the number of patients amenable to gene therapy and make some patients candidates for retreatment when necessary 70 Acceleration and Augmentation of Femoral Segmental Bone Healing by Adipose-Derived Stem Cells Engineered with Hybrid Baculovirus Vectors Conferring Sustained Transgene Expression Chin-Yu Lin,1 Kun-Ju Lin,2 Chun-Yu Kao,1 Tzu-Chen Yen,2 YuHan Chang,3 Yu-Chen Hu.1 Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan; 2Nuclear Medicine and Molecular Imaging Center, Chang Gung Memorial Hospital, Taoyuan, Taiwan; 3Orthopaedic, Chang Gung Memorial Hospital, Taoyuan, Taiwan Massive segmental defects arising from trauma or tumor resection remain a challenging clinical problem To heal massive, segmental bone defects using adipose-derived stem cells (ASCs), which alone cannot heal large defects, we hypothesized that sustained expression of factors promoting bone regeneration (BMP2) and angiogenesis (VEGF) provides continuous stimuli to augment the healing Insect baculovirus (BV) holds promise for gene therapy and efficiently transduces stem cells, but it only mediates transient transgene expression Therefore we developed a dual BV system whereby one BV expressed FLP recombinase (BacFLP) while the other hybrid BV harbored an Frt-flanking transgene cassette The New Zealand White (NZW) rabbit ASCs were transduced with the BacFLP and hybrid BV or conventional BV, seeded to scaffolds and transplanted into critical-sized (10 mm) femoral segmental defects in NZW rabbits The bone regeneration was assessed by radiography, Positron Emission Tomography/Computer Tomography (PET/CT) at 2, 4, and 12 weeks post-transplantation (wpt), and µCT, histological staining, biomechanical torsional testing at 12 wpt, respectively Within the ASCs transduced with BacFLP and the hybrid BV, the transduction efficiency reached 98% while the FLP/Frt-mediated recombination efficiency approached 46%, leading to cassette excision off the BV genome, enabling transgene persistence in episomal form and prolonging the expression to >28 days Transduction of ASCs with the BMP2-encoding hybrid BV led to prolonged BMP2 expression and augmented ASCs osteogenesis even without other osteogenic supplements ASCs engineered by conventional BV transiently expressing BMP2/VEGF (S group) only healed the critical-sized femoral bone defects in 40% of NZW rabbits at 12 wpt, but ASCs engineered by the hybrid vectors persistently expressing BMP2/VEGF (L group) healed the critical-sized defects in 12 out of 12 animals in weeks Compared with the S group, the L group not only accelerated the healing, but also ameliorated the bone metabolism, bone volume, bone density, angiogenesis and mechanical properties These data confirmed our hypothesis that persistent BMP2/VEGF expression is essential Use of the hybrid BV vector for ASCs engineering represents a novel approach to treating massive segmental bone defects necessitating concerted ossification and vascularization 71 Restoration of Dystrophin Expression in the mdx Mouse Model for Duchenne Muscular Dystrophy (DMD) Induced by RTC13 Refik Kayali,1 Liutao Du,2 Jin-Mo Ku,3 Gladys Completo,3 Olga Prikhodko,1 Yosuf Subat,1 Hailiang Hu,2 Michael Jung,3 Richard A Gatti,2 Carmen Bertoni.1 Department of Neurology, David Geffen School of Medicine University of California Los Angeles, Los Angeles, CA; Department of Pathology and Laboratory Medicine, David Geffen School of Medicine University of California Los Angeles, Los Angeles, CA; 3Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA Molecules that induce ribosomal read-through of nonsense mutations in mRNA and allow production of a full-length functional protein hold great therapeutic potential for the treatment of many genetic disorders Two such read-through compounds, RTC13 S28 Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy 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 ... 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... 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... 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