251. rAAV-Mediated Gene Therapy To Treat Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D)
Authors: Christina A. Pacak, Denise Cloutier, Irene Zolotukhin, Gabriel S. Gaidosh, Kevin Campbell, Glenn A. Walter, Barry J. Byrne
Journal: Molecular Therapy (2004)

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The long-term goal of this project is to develop a clinically relevant gene therapy approach for the treatment of limb girdle muscular dystrophy type 2D (LGMD-2D). LGMD-2D is the result of mutations in the alpha sarcoglycan (ASG) gene and is characterized by the progressive development of lesions in skeletal muscle due to deterioration of the sarcolemma. Our initial objective has been to demonstrate that recombinant adeno-associated virus (rAAV) can be used as a vehicle for delivery of the ASG gene to dystrophic muscle. We first undertook an in vitro promoter comparison study in which we determined that AAV serotype 1 with both the CMV and a modified form of the murine creatine kinase promoter that confers muscle specific expression, tMCK, have the ability to drive ASG gene expression in differentiated ASG-/- myoblasts at nearly equal levels. To evaluate gene delivery in vivo we have developed a non-invasive imaging assay using MRI that enables us to locate and measure the random development of lesions within skeletal muscle of ASG-/- mice. 1 and 2-month-old ASG -/- mice were imaged to determine the initial size and location of lesions in muscles of the lower extremities. Less than 15% of the tibialis anterior (TA) muscles from mice imaged at 1 month and approximately 90% of TA muscles imaged at 2 months showed lesion development. Therefore, we have established that there is a critical window for prevention and/or correction of lesion development in specific muscles during the first few weeks of life in the ASG-/- mouse. Both age groups were injected in one TA with 1 × 1011 viral particles of either rAAV1-tMCK-ASG or rAAV2-CMV-ASG. For 4 months, monthly MRI was performed to observe lesion development. These images have enabled us to generate T2 maps to quantify areas of elevated intensity in specific regions of interest. Upon sacrifice, we have performed force mechanics to evaluate the ability of our therapy to provide not only physiological but also functional correction to dystrophic muscle. We have used immunohistochemistry to determine ASG expression and Evans Blue Dye (EBD) to elucidate the presence of lesions on tissue sections and thereby further confirm the MRI data. Tri-chrome staining was performed to identify lesions that have become infiltrated with collagen. Initial MRI, immunohistochemistry and force mechanics data indicate that each vector is able to confer both physiological and functional correction in the ASG-/- mouse model however, in both cases; the majority of transgene expression was detected along the needle path of the injection site. In a related experiment,<12 hour old ASG-/- pup legs have been injected with 1 × 1011 viral particles of either AAV1-tMCK-ASG or AAV1-tMCK-LacZ to determine if better vector distribution (and ultimately more successful correction) can be obtained when therapy is delivered at an earlier point in development. In conclusion, our preliminary data suggests that (rAAV) is an effective vehicle for delivery of the ASG gene in ASG-/- skeletal muscle and is therefore a promising vector for gene delivery in vivo but time of injection and vector distribution may be critical factors in successful treatment.