Systemic Delivery of the Interleukin-1 Receptor Antagonist Protein Using a New Strategy of Direct Adenoviral-Mediated Gene Transfer to Skeletal Muscle Capillary Endothelium in the Isolated Rat Hindlimb

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 40% of total body weight; it possesses a high density, accessible capillary network that is resistant to injury and thus may be a logical target for adenoviral vectors. We 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-1ra) was infused and subsequently flushed from the circulation after a 30-min dwell period. Gene transfer over a 10⁹–10¹² particle/ml range to the gastrocnemius capillary endothelium and muscle fibers was highly efficient and titer-dependent, reaching maximum transduction rates of 71 ± 7% and 25 ± 5%, respectively, 5 days after gene transfer (n = 3–8 rats/group, p < 0.05). hpAP transgene expression was barely detectable at 14 days. No 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-1ra had 241 ± 66 pg IL-1ra/mg protein at 5 days, while those given Ad-hpAP, negative control (n = 3) had 35 ± 14 pg IL-1ra/mg protein (p < 0.05). Ad-IL-1ra rats (n = 4) had serum levels of 185 ± 20 pg/ml IL-1ra at 5 days whereas Ad-hpAP control rats (n = 5) had no IL-1ra detectable (p < 0.0001). Athymic rats given Ad-IL-1ra (n = 6) had serum levels of 493 ± 62 pg/ml IL-1ra 14 days after transduction, and IL-1ra was detected for up to 98 days. Sera from Ad-IL-1ra athymic rats significantly inhibited IL-1β-induced (1 ng/ml) prostaglandin E₂ (PGE₂) 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-1ra. 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.

Overview summary

Here we report highly efficient adenoviral-mediated gene transfer to the rat skeletal muscle capillary endothelium and muscle fibers during vascular isolation of the hindlimb. Gene transfer over a 10⁹–10¹² particle/ml range to the gastrocnemius capillary endothelium and muscle fibers is highly efficient and titer dependent, reaching maximum transduction rates in these tissue of 71 ± 7% and 25 ± 5%, respectively, 5 days after gene transfer. Adenovirus-induced inflammation in the skeletal muscle was not observed and distant organ gene transfer was minimal or absent. When adenoviral interleukin-1 receptor antagonist (Ad-IL-1ra) was used to transduce the gastrocnemius capillary endothelium and muscle fibers, serum IL-1ra levels were 185 ± 20 pg/ml. Athymic rats given Ad-IL-1ra had serum levels of 493 ± 62 pg/ml IL-1ra 14 days after transduction and IL-1ra was detected for up to 98 days. This gene transfer strategy results in substantial transduction of capillary endothelium as well as skeletal muscle fibers sufficient to achieve pharmacologic levels of recombinant protein without evidence of local tissue injury.