Optimization of a novel low-stength electroporation system for ex vivio gene transfer in human heart

Abstract

Listen

Translate article

Electroporation (EP) is commonly used for in vitro gene transfection of cell lines and primary cultures, by applying short duration, high intensity (200-2000 volts/cm) electric field pulses to cells. Recently, we invented a novel low-strength EP system for ex vivo gene transfer in human heart. We developed a device consisted of two electrode arrays directly in contact with the endocardium and epicardium in order to achieve uniform gene transfer to the entire adult human heart. To optimize the EP parameters for achieving maximum gene transfer efficiency and lowest cardiac adverse effect, the human explanted hearts from patients with end-stage heart failure underwent orthotopic heart transplantation were used. The heart was mounted on a customized Langendorff perfusion apparatus, then human recombinant IL-10 gene was infused through intracoronary. The EP was applied during gene infusion for 20 minutes. With 10 volt/cm EP field strength, the maximum gene transfer efficiency was achieved, but associated with a mild cardiac contractility reduction in 15% of hearts. However, with 1-5 volt/cm electric field strength, no cardiac adverse effects was observed. The optimized pulse length was 1-10 ms, and the optimized number of pulse 1-20. The burst-interval >2 min prevented myocytes damage. With this optimized EP, the homogeneous electropermeabilization in whole heart was observed within 3 minuets after the beginning of EP using propidium iodide, a membrane-impermeant fluorescent probe. A significant increase in IL-10 mRNA level in myocardium of both ventricles was detectable in 2 hours and to 2.1±0.2 fold in 10 hours by RT-PCR and the fluorescent image of Green Fluorescence Protein expression was able to be observed within 8 hours. In conclusion: these results suggest that this new EP-mediated gene transfer technique may be potentially applicable for gene delivery in human heart.