Electroporation‐mediated delivery of a naked DNA plasmid expressing VEGF to the porcine heart enhances protein expression

Department of Surgery, University of South Florida, Tampa, FL, USA.
Gene therapy (Impact Factor: 3.1). 12/2009; 17(3):419-23. DOI: 10.1038/gt.2009.153
Source: PubMed


Gene therapy is an attractive method for the treatment of cardiovascular disease. However, using current strategies, induction of gene expression at therapeutic levels is often inefficient. In this study, we show a novel electroporation (EP) method to enhance the delivery of a plasmid expressing an angiogenic growth factor (vascular endothelial growth factor, VEGF), which is a molecule previously documented to stimulate revascularization in coronary artery disease. DNA expression plasmids were delivered in vivo to the porcine heart with or without coadministered EP to determine the potential effect of electrically mediated delivery. The results showed that plasmid delivery through EP significantly increased cardiac expression of VEGF compared with injection of plasmid alone. This is the first report showing successful intracardiac delivery, through in vivo EP, of a protein expressing plasmid in a large animal.

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Available from: Bernadette Ferraro,
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    • "So far, several methods that can induce transfection of naked pDNA into cardiac tissue have been reported. Direct myocardial injection of naked pDNA has been used for gene therapy for heart failure [19], [20]; retrograde injection of naked pDNA in the coronary sinus by catheterization was used for whole cardiac gene transfer [21], and in vivo electroporation was conducted on rat and porcine hearts [22], [23]. As shown in Fig. 4C, the transfection efficiency by tissue suction in the liver was equal to that of the liver pressure-mediated transfection method. "
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    ABSTRACT: We have developed an in vivo transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.
    PLoS ONE 07/2012; 7(7):e41319. DOI:10.1371/journal.pone.0041319 · 3.23 Impact Factor
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    • "EP was originally discovered for the delivery of plasmid DNA into cells in vitro by Neumann et al. in 1982.7 Since the first report of successful gene electrotransfection of the skin,8 the method has been increasingly used for the delivery of different types of DNA/RNA molecules into cells in vitro and different tissues, including muscle and tumors in vivo.9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 Furthermore, gene electrotransfer has also proved to be safe and effective in the treatment of cancer. A report on the first clinical study of gene electrotransfer, which used therapeutic gene coding for interleukin-12 for the treatment of metastatic melanoma, has already been published by Daud et al.20 There are also other clinical studies in progress on muscle tissue for DNA vaccination, systemic delivery of therapeutic proteins and correction of gene defects in muscles; therefore, the studies on muscle cell cultures and muscle tissue are of special importance. "
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    ABSTRACT: Electropermeabilization (EP) is an effective method of gene transfer into different tissues. During EP, reactive oxygen species (ROS) are formed, which could affect transfection efficiency. The role of generated ROS and the role of antioxidants in electrotransfer in myoblasts in vitro and in Musculus tibialis cranialis in mice were, therefore, investigated. We demonstrate in the study that during EP of C2C12 myoblasts, ROS are generated on the surface of the cells, which do not induce long-term genomic DNA damage. Plasmid DNA for transfection (pEGFP-N1), which is present outside the cells during EP, neutralizes the generated ROS. The ROS generation is proportional to the amplitude of the electric pulses and can be scavenged by antioxidants, such as vitamin C or tempol. When antioxidants were used during gene electrotransfer, the transfection efficiency of C2C12 myoblasts was statistically significantly increased 1.6-fold with tempol. Also in vivo, the transfection efficiency of M. tibialis cranialis in mice was statistically significantly increased 1.4-fold by tempol. The study indicates that ROS are generated on cells during EP and can be scavenged by antioxidants. Specifically, tempol can be used to improve gene electrotransfer into the muscle and possibly also to other tissues.
    Gene therapy 06/2011; 19(3):312-20. DOI:10.1038/gt.2011.97 · 3.10 Impact Factor
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    • "Similarly in the mice heterotopic heart transplant model Wang et al [16] demonstrated that ex vivo electroporation mediated gene transfer of the graft before its implantation, allowed significant gene expression. Very recently this technique was also applied on the pig heart [23]. Therefore, it was with no doubt that electroporation would enhance gene expression in the cardiac muscle. "
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    ABSTRACT: Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase) and TNT (Troponin T) were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.
    PLoS ONE 12/2010; 5(12):e14467. DOI:10.1371/journal.pone.0014467 · 3.23 Impact Factor
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