296. Expression of FKRP Consequently Restored Functional Glycosylation of α-DG in the Skeletal and Cardiac Muscles in FKRP Mutant Mice

MUSCULO-SKELETAL GENE & CELL THERAPY II would benefit from RNAi-mediated reduction of the pathogenic alleles underlying their disease. In this study, we developed the first RNAi-based, pre-clinical treatment for LGMD1A, caused by dominant mutation in one allele of the myotilin (MYOT) gene. Our strategy involved engineering MYOT-targeted artificial microRNA (miMYOT) vectors to knockdown mutant MYOT in muscles of an LGMD1A mouse model. Three months after treatment, we found miMYOT vectors, but not controls, significantly reduced soluble mutant MYOT protein to undetectable levels, and the protein aggregates, one of the characteristics of LGMD1A, were either absent or very small in treated muscles. Our miMYOT vectors also significantly improved muscle mass and myopathic histopathology, such as reduced centrally localized nuclei and increased myofiber diameters. Furthermore, miMYOT vectors significantly improved muscle functions including specific and absolute forces in affected muscles after nine-month treatment. These studies represent important first steps toward translating targeted RNAi gene therapy approaches for LGMD1A, and our RNAi strategies could be adapted to impact a large class of dominant muscle disorders, including other forms of LGMD.

OA. Combination of cell therapy and growth factor gene therapy may further enhance this response. This study evaluated rAAV5IGF-I transduced chondrocytes for repair of full-thickness chondral defects in the equine femoropatellar joint model. Methods: A scAAV vector with equine IGF-1 or eGFP was developed with CMV promoter, and propagated at Childrens Hospital of Philadelphia. Autogenous chondrocytes were isolated and infected with 10e5 AAV vg/cell expressing GFP (8 horses), IGF-1 (8 horses), or remained as uninfected chondrocytes (8 horses). Defects (15mm diameter) in the lateral trochlear ridges of the left and right femur were repaired with arthroscopic implantation of gene enhanced or naive chondrocytes in fibrin. Healing was assessed at 8 months by MRI, gross pathology, histology and immunohistochemistry, and gene expression. Results: Arthroscopic implantation of autologous chondrocytes transduced with rAAV5-IGF-I resulted in significantly better total gross healing scores at 8 months, compared to paired control defects (p=0.008).

296. Expression of FKRP Consequently Restored Functional Glycosylation of α-DG in the Skeletal and Cardiac Muscles in FKRP Mutant Mice

Qi Long Lu,1 Lei Xu,1 Pei Juan Lu,1 Chi-Hsien Wang,2 Elizabeth Keramaris,1 Chunping Qiao,2 Bin Xiao,2 Derek J. Blake,3 Xiao Xiao.2 1 McColl-Lockwood Laboratory for Muscular Dystrophy Research, Carolinas Medical Center, Charlotte, NC; 2Division of Molecular Pharmaceutics, University of North Carolinas at Chapel Hill, Chapel Hill, NC; 3Department of Psychological Medicine, Cardiff University, Cardiff, United Kingdom.

Mutations in FKRP gene are associated with a wide range of muscular dystrophies from mild limb-girdle muscular dystrophy (LGMD) 2I to severe Walker-Warburg syndrome (WWS) and muscleeye-brain disease (MEB). The characteristic biochemical feature of these diseases is the hypoglycosylation of -dystroglycan (-DG) in muscles. Currently there is no effective treatment available. In this study we examined the therapeutic effects of systemic FKRP gene delivery by Adeno-associated virus serotype 9 vector (AAV9) in the FKRP mutant mouse model with a proline to leucine missense mutation (P448L). Our results showed that systemic gene delivery resulted in FKRP expression in all striated muscles examined with the highest levels in cardiac muscle. Consistent to our previous observation, FKRP protein localized in Golgi apparatus in myofibers. Expression of FKRP consequently restored functional glycosylation of -DG in the skeletal and cardiac muscles. Significant improvement in dystrophic pathology, serum creatine kinase levels and muscle function was observed. Only limited FKRP transgene expression was detected in kidney and liver with no detectable toxicity. Our results provided evidence for applicability of AAV-mediated gene replacement therapy for FKRP-related muscular dystrophies.

297. Minimally Invasive Implantation of Autologous Chondrocytes Transduced with rAAV5-IGF-I Improves Longterm Cartilage Repair in Full-Thickness Chondral Defects in the Equine Model Kyla Ortved,1 Alan J. Nixon.1 1 Comparative Orthopaedics Laboratory, Cornell University, Ithaca, NY.

Introduction: Articular cartilage injury occurs frequently and often precipitates osteoarthritis. Autologous chondrocyte implantation (ACI) is being used more extensively to return function and minimize Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

Additionally, defects repaired with rAAV5-IGF-I transduced chondrocytes had significantly better healing scores than defects repaired with chondrocytes alone (p=0.02). Mean IGF-I concentration in synovial fluid from rAAV5-IGF-I treated joints (1.99ug/ml) was significantly increased over control joints (1.46ug/ml) at 7 days postoperatively (p=0.03).

Semiquantitative analysis of histology scores indicated improved cartilage maturity and several constituents in AAV-IGF-1 implanted defects. Discussion: Arthroscopic implantation of autologous chondrocytes transduced with rAAV5-IGF-I improved healing of full-thickness chondral defects at 8 weeks and 8 months postimplantation compared to defects filled with chondrocytes or fibrin alone. rAAV-IGF-I transduced cells produced substantial IGF-I ligand that increased IGF-I concentrations in the synovial fluid compared to pre-implant levels. This study provides evidence that genetically modified chondrocytes can improve healing of articular cartilage in a challenging cartilage repair model. Significance: Arthroscopically implanted autologous chondrocytes over-expressing IGF-I improve cartilage repair which may be further enhanced by integrating vectors and concurrent cytokine suppression. Acknowledgements: Study funded by NIH grant 5R01-AR055373-02. S113