73. Inhibition of CD26 Activity Enhances Engraftment of Donor Cells to Regenerating and Dystrophic Skeletal Muscle

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 1 Genethon, Evry, France; 2INSERM UMR 649, Nantes, France; 3 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 8 (rAAV8) expressing a modified U7 snRNA specific for the skipping of exons 5 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 Française 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 1 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 do 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 9 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 1 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 9 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