Gene Transfer to Intact Mesenteric Arteries by Electroporation

Abstract

The purpose of the present study was to develop a rapid, reproducible method of non-viral gene transfer to the intact vasculature. Male Sprague-Dawley rats were anesthetized, a midline abdominal incision was made and segmental branches of the superior mesenteric artery were dissected free of surrounding mesentery. A specially designed electroporation probe was placed around the neurovascular bundle and the electroporation chamber filled with a solution containing the firefly luciferase expressing plasmid (pCMV-luxDTS) or the green fluorescent protein expressing plasmid (pEGFP-N1). Vessels were electroporated with eight 10 msec pulses of 200V/cm. Sixty seconds after electroporation, the DNA solution was removed, the intestine returned to the abdomen and the abdominal wall closed with suture and metal wound clips. Six hours to 5 days later, rats were sacrificed and electroporated vessels were recovered. Luciferase activity of the blood vessels was monitored. Gene expression was detected as early as 6 h postelectroporation, peaked at 1-3 days with levels up to 1 ng of reporter gene per vessel segment and returned towards baseline by day 5. Histological analysis of blood vessel segments revealed GFP positive cells throughout the thickness of the vessel wall (endothelial cells to adventitia). Responses of electroporated vessels to vasoconstricting stimuli were indistinguishable from those of control vessels at either 2 or 40 days post-treatment. The results of this study provide evidence that electroporation is an effective means for introducing naked DNA into the blood vessel wall and form the basis for future studies on targeted gene therapy to the intact vasculature.

Ultrasound-assisted non-viral gene transfer to the salivary glands

“…However, delivery of non-viral vectors to target tissues, such as the salivary gland, is extremely challenging as eukaryotic cell membranes typically exclude genetic material. Techniques such as hydrodynamic gene delivery, 13 various nanoparticles and electroporation [14][15][16] have shown proof of the principle in selective applications, but have not been used successfully to achieve gene transfer to the salivary gland.…”

Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature

“…16 This technique results in high-level gene expression in all cell layers of the vasculature and results in no tissue damage or downstream ischemia. 16 Since the cells of the vasculature are terminally differentiated and nondividing, this model serves as an excellent system in which to study the effects of the SV40 DTS.…”

“…16 This technique results in high-level gene expression in all cell layers of the vasculature and results in no tissue damage or downstream ischemia. 16 Since the cells of the vasculature are terminally differentiated and nondividing, this model serves as an excellent system in which to study the effects of the SV40 DTS. In this study, we demonstrate that incorporation of the SV40 DTS into expression plasmids results in 10-40-fold increases in vascular gene expression, confirming the function of DNA nuclear targeting sequences in vivo.…”

“…16 This procedure is rapid, safe, and results in no ischemic damage to the treated vessels or downstream tissues. However, it is possible that the application of the electric field or the procedure itself could promote cell division or neovascularization, thus complicating our analysis.…”

“…Vessels were electroporated as previously described. 16 Briefly, male SpragueDawley rats (200-400 g) were anesthetized with isoflurane, a midline incision was made, and the small intestine was exteriorized. Approximately 1 cm of each neurovascular bundle was placed individually in the electrode and covered with a solution of 10 mM Tris, pH 8, 1 mM EDTA, 140 mM NaCl.…”