Gene Transfer to Skeletal Muscle 1797

Current gene therapy strategies using adenoviral vectors to target the lung or liver have been complicated by an acute inflammatory response that can result in loss of transgene expression as well as tissue injury and necrosis. Skeletal muscle comprises 4 0 % oftotal body weight; it possesses a high density, accessible capillary network that is resistant to injury and thus m a y be a logical target for adenoviral vectors. W e hypothesized that adenoviral transduction of the rat skeletal muscle capillary bed during vascular isolation would achieve efficient gene transfer sufficient to achieve systemic serum levels of a recombinant protein without significant tissue injury. During vascular isolation of the hindleg, a replication-incompetent adenovirus (Ad) encoding for either the marker gene, human placental alkaline phosphatase (hpAP), or interleukin-1 receptor antagonist (IL-lra) was infused and subsequently flushed from the circulation after a 30-min dwell period. Gene transfer over a 10^-10^^ particle/ml range to the gastrocnemius capillary endothelium and muscle fibers was highly efficient and titer-dependent, reaching m a x i m u m transduction rates of 71 ± 7 % and 25 ± 5 % , respectively, 5 days after gene transfer (n = 3-8 rats/group, p < 0.05). h p A P transgene expression was barely detectable at 14 days. N o significant tissue injury or necrosis of the skeletal muscle was observed at 5 and 14 days, and distant organ gene transfer was minimal or absent. Gastrocnemius muscle from rats (n = 4) given Ad-IL-lra had 241 ± 66 pg IL-lra/mg protein at 5 days, while those given Ad-hpAP, negative control (n = 3) had 35 ± 14 pg IL-lra/mg protein (p < 0.05). Ad-IL-lra rats (« = 4) had serum levels of 185 ± 20 pg/ml IL-lra at 5 days whereas Ad-hpAP control rats (« = 5) had no IL-lra detectable (p < 0.0001). Athymic rats given Ad-IL-lra (n = 6) had serum levels of 493 ± 62 pg/ml IL-lra 14 days after transduction, and IL-lra was detected for up to 98 days. Sera from Ad-IL-lra athymic rats significantly inhibited IL-l/3-induced (1 ng/ml) prostaglandin Ej (PGEi) production from cultured endothelial cells by 82 ± 2 % (p < 0.001). Thus, this gene transfer strategy is the first to result in substantial transduction of both skeletal muscle capillary endothelium and fibers, sufficient to achieve pharmacologic levels of IL-lra. Although no acute tissue injury or necrosis was observed, persistence of transgene expression in athymic rats suggests that loss of expression in normal rats was by an immune-mediated mechanism. O V E R V I E W S U M M A R Y Gene transfer over a lO'-lQi^ particle/ml range to the gastrocnemius capillary endothelium and muscle fibers is Here we report highly efficient adenoviral-mediated gene highly efficient and titer dependent, reaching maximum transfer to the rat skeletal muscle capillary endothelium and transduction rates in these tissue of 71 ± 7 % and 25 ± 5%, muscle fibers during vascular isolation of the hindlimb. respectively, 5 days after gene transfer. Adenovirus-induced Departments of 'Surgery, ̂ Pathology, and ̂Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109. "Department of Intemal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242. D̂epartment of Surgery, University of California, San Francisco, San Francisco, CA 94143.