The purpose of this study was to assess the behavior of pseudotyped recombinant adeno-associated virus type 1 (rAAV2/1) vector genomes in dystrophic skeletal muscle. A comparison was made between a therapeutic vector and a reporter vector by injecting the hindlimb in a mouse model of Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) prior to disease onset. We hypothesized that the therapeutic vector would establish long-term persistence through prevention of myofiber turnover. In contrast, the reporter vector genome copy number would diminish over time due to disease-associated muscle degradation. One day old alpha sarcoglycan knockout mice (sgca-/-) were injected with 1 × 1011 vector genomes of rAAV2/ 1-tMCK-sgca in one hindlimb and the same dose of rAAV2/1-tMCK-LacZ in the contra lateral hindlimb. Newborn mice are tolerant of the foreign transgene allowing for long-term expression of both the marker and the therapeutic gene in the null background. At 2 time-points following vector administration, hindlimb muscles were harvested and analyzed for LacZ or sarcoglycan expression. Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene. We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer. In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss. Mitigating the membrane instability inherent in dystrophic muscle was able to prolong the life of individual myofibers.
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We would like to express our gratitude to Dr. Kevin P. Campbell from the University of Iowa for providing the sgca-/-mouse model used in these studies. We would also like to acknowledge the University of Florida Powell Gene Therapy Center Toxicology staff for vector genome quantification and the Powell Gene Therapy Center Viral Vector Core for making the AAV used in these experiments. This work was supported in part by an American Heart Association Pre-doctoral Fellowship Award-Florida and Puerto Rico Affiliate (to CAP), the NIH National Heart, Lung, and Blood Institute grant PO1 HL59412; National Institute of Diabetes and Digestive and Kidney Diseases grant PO1 DK58327; AT-NHLBI-U01 HL69748; and the AHA National Center (to C.S.M.).