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 polyvinylpyrrolidone (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.

Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage

“…Treated animals were killed 1 week after the experiments as peak levels of transgene expression in muscle were usually found between 1 and 2 weeks. 18,19 Muscles were removed and sections were cut at intervals of 100 mm. The number of GFP-positive fibres was counted.…”

Intratumoral delivery of IL-12 gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity

“…27 Recently, intra-muscular expression of biologically active human IGF-I and factor IX have also been demonstrated in vivo using this system. 28,29 To assess the immunogenicity of plasmid formulated in PINC, we assayed for the presence of anti-plasmid antibodies in serum of dogs repeatedly injected with this formulation and did not detect any antibodies to the administered plasmid (unpublished data). Furthermore, we observed that delivery of our expression system for IL-12 lacks any obvious toxicity such as loss of weight or any signs of distress, that has been reported in mice following systemic delivery of IL-12 protein.…”

“…27 This system has been used to obtain significant biological activity of a variety of transgene products. 28,29 Using this system to deliver IL-12 into two tumor models -immunogenic (Renca) and less immunogenic (CT26) -we show that up to 50% of tumorbearing mice in both models reject their tumors with the help of a primarily CD8 + T cell-mediated immune response that has long lasting memory.…”

Skeletal muscle regeneration after insulin-like growth factor I gene transfer by recombinant Sendai virus vector

“…These levels of hIGF-I protein production by the L6 cell line infected with the hIGF-I/SeV were over 100-fold higher than those reported for the C2C12 cell line tranfected with a naked plasmid. 18 (3) The L6 cell line is known to not express IGF-I but present IGF-I receptor. 19 Therefore, the cell line is responsive to an exogenous IGF-I, thus resulting in the induction of hypertrophy.…”