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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).

Naked plasmid DNA (pDNA)-based gene therapy has low delivery efficiency, and consequently, low therapeutic effect. We present a biodegradable nonionic triblock copolymer, PEG(13)-PLGA(10)-PEG(13), to enhance gene delivery efficiency in skeletal muscle. Effects of PEG(13)-PLGA(10)-PEG(13) on physicochemical properties of pDNA were evaluated by atomic force microscopy (AFM) imaging, gel electrophoresis and zeta-potential analysis. AFM imaging suggested a slightly compacted structure of pDNA when it was mixed with the polymer, while zeta-potential measurement indicated an increased surface potential of negatively charged pDNA. PEG(13)-PLGA(10)-PEG(13) showed a relatively lower toxicity compared to Pluronic P85 in a skeletal muscle cell line. The luciferase expression of pDNA delivered in 0.25% polymer solution was up to three orders of magnitude more than branched polyethylenimine (bPEI(25 k))/pDNA and three times more than that of naked pDNA five days after intramuscular administration. This in vivo gene delivery enhancement was also observed displaying a two-fold higher expression of human vascular endothelial growth factor (VEGF). Based on fluorescence labeled pDNA distribution, it is speculated that the greater diffusivity of PEG(13)-PLGA(10)-PEG(13)/pDNA compared to bPEI(25 k)/pDNA accounts for better transfection efficiency in vivo. To summarize, combining PEG(13)-PLGA(10)-PEG(13) with pDNA possesses the potential to improve gene delivery efficiency in skeletal muscle.

Delivery of the hypoxia-inducible vascular endothelial growth factor (RTP-VEGF) plasmid using a novel reducible disulfide poly(amido ethylenediamine) (SS-PAED) polymer carrier was studied in vitro and in vivo. In vitro transfection of primary rat cardiomyoblasts (H9C2) showed SS-PAED at a weighted ratio of 12:1 (polymer/DNA) mediates 16 fold higher expression of luciferase compared to an optimized bPEI control. FACS analysis revealed up to 57+/-2% GFP positive H9C2s. The efficiency of plasmid delivery to H9C2 using SS-PAED was found to depend upon glutathione (GSH) levels inside the cell. SS-PAED mediated delivery of RTP-VEGF plasmid produced significantly higher levels of VEGF expression (up to 76 fold) under hypoxic conditions compared to normoxic conditions in both H9C2 and rat aortic smooth muscle cells (A7R5). Using SS-PAED, delivery of RTP-VEGF was investigated in a rabbit myocardial infarct model using 100 mug RTP-VEGF. Results showed up to 4 fold increase in VEGF protein expression in the region of the infarct compared to injections of SS-PAED/RTP-Luc. In conclusion, SS-PAED mediated therapeutic delivery improves the efficacy of ischemia-inducible VEGF gene therapy both in vitro and in vivo and therefore, has potential for the promotion of neo-vascular formation and improvement of tissue function in ischemic myocardium.

Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (Flt-1, KDR/flk-1) have become critical for anti-tumor angiogenesis therapy. Previously, we synthesized PEI-g-PEG-RGD conjugate and evaluated as an angiogenic endothelial polymeric gene carrier. In this study, PEI-g-PEG-RGD/pCMV-sFlt-1 complexes are evaluated in terms of tumor growth inhibition in vivo. Complexes were repeatedly injected systemically via tail vein into subcutaneous tumor-bearing mice. As a result, tumor growth was inhibited in the PEI-g-PEG-RGD/pCMV-sFlt-1 injected group. However, this effect was not identified in PEI-g-PEG/pCMV-sFlt-1 or PEI-g-PEG-RGD/pCMV-GFP control groups. Moreover, the survival rate increased in the PEI-g-PEG-RGD/pCMV-sFlt-1 group compared with the controls group. These results suggest that delivery of pCMV-sFlt-1 using PEG-g-PEG-RGD may be effective for anti-angiogenic gene therapy.