Expression of Biologically Active Human Insulin-like Growth Factor-I Following Intramuscular Injection of a Formulated Plasmid in Rats

Abstract

Recent evidence has shown that insulin-like growth factor-I (IGF-I) plays an important role in the development, maintenance, and regeneration of peripheral nerves and skeletal muscle. IGF-I offers the potential to treat neuromuscular diseases in humans. We have developed a nonviral gene therapy method to express and produce localized and sustained therapeutic levels of IGF-I within target muscles by intramuscular injection of formulated plasmids. The purpose of the present study was to demonstrate that intramuscular injection of a plasmid encoding human IGF-I (hIGF-I) and engineered to restrict expression to skeletal muscle produces sustained local concentrations of biologically active hIGF-I. Normal rats received a single intramuscular injection of plasmids formulated as a complex with polyvinypyrrolidone (PVP). Results show that hIGF-I mRNA and hIGF-I protein were detectable in the injected muscles for the duration of the study (28 days), whereas the hIGF-I protein was not detected in blood. Biological activity of hIGF-I was determined by immunodetection of a nerve-specific growth-associated protein, GAP-43, an indicator of motor neuron sprouting. Placement of human growth hormone (hGH) 3′ untranslated region enhanced GAP-43 staining, probably due to improved secretion of hIGF-I. Enhanced immunoreactivity of GAP-43 was observed in muscles injected with the formulated hIGF-I plasmid when compared to controls. These results demonstrate that intramuscular injection of hIGF-I plasmid formulated as a complex with PVP produces a localized and sustained level of biologically active hIGF-I. Alila et al. showed that intramuscular injection of a plasmid encoding human insulin-like growth factor-I (hIGF-I), and formulated as a complex with polyvinylpyrrolidone (PVP) produces local concentrations of biologically active hIGF-I. Chicken skeletal α-actin promoter was used to restrict expression of hIGF-I to skeletal muscle cells. Placement of hGH 3′ untranslated region enhanced biological activity of the formulated plasmid, measured by GAP-43 immunostaining. Measurement of hIGF-I mRNA and hIGF-I protein in muscle tissues showed that expression lasted for 28 days. Neither hIGF-I protein nor antibodies to hIGF-I was detectable in blood.