Efficient expression of vascular endothelial growth factor using minicircle DNA for angiogenic gene therapy.

The application of plasmid DNA (pDNA)-based gene therapy is limited by its inefficient transgene expression. In this study, minicircle DNA was evaluated for efficient vascular endothelial growth factor (VEGF) expression in skeletal muscle cells. Production of minicircle DNA encoding VEGF was studied by a semi-quantitative electrophoresis method leading to optimized bacterial culture conditions and producing high purity (86.6%) minicircle DNA. The VEGF minicircle DNA under control of the SV40 promoter (pMini-SV-VEGF) showed an increased amount of VEGF mRNA and up to 8 times more VEGF expression than a conventional plasmid (pSV-VEGF) in two different skeletal muscle cell lines (C2C12 and L8). Minicircle DNA with different promoters, including the SV40, CMV and chicken beta-actin, was tested for VEGF expression in a C2C12 skeletal muscle cell line. The high VEGF expression generated by minicircle DNA stimulated efficient endothelial cell growth in vitro. Furthermore, minicircle DNA expressed higher VEGF compared to conventional plasmid in the tibialis anterior (TA) muscle of mice. Taken together, the results suggest that minicircle DNA is an efficacious gene vector for angiogenic VEGF expression in skeletal muscle and may be useful for treating peripheral arterial disease (PAD).