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Deb K. Mojumder, Theodore G. Wensel, Stephen C. Pflugfelder; Optimizing Electrically Assisted Delivery Of Expression Vectors To The Cornea And Iris. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1407.
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Gene therapy involves the targeting & expression of expression vectors in the desired cell-type of interest. Electrically assisted gene delivery achieves this by using pulses of electrical energy which causes electro-permeabalization of the cell membrane and electrophoresis of charged expression-vectors towards the cell-type of interest. Optimizing electropermeabalization and electrophoresis while using minimum amount of electrical energy results in less tissue damage. Although cornea is the most approachable part of the eye, expressing genes by various techniques have been inefficient (Hao, J., S. K. Li, et al. Brain Res Bull 81(2-3): 256-61.).
Plasmids encoding eGFP fluorescent proteins driven by a CMV promoter were purified by using endotoxin-free Qiagen maxipreps. Topical application or anterior-chamber injections of plasmids were performed in age-matched adult-mice (6-8weeks old) followed by electroporation with a custom built personal computer controlled electroporator. Corneal whole mounts were performed to examine fluorescent protein expression.
We discovered that expression vectors (pEGFP-N1 vector; 0.3 to 10 µg/µl) injected into the anterior chamber followed by electroporation using square-wave pulses (Voltage: 100V; Pulse width: 50ms; Interpulse interval: 850ms) oriented such that the direction of electrophoresis of the expression vectors was towards the cornea, there was a high level expression of plasmids in the corneal stroma corneal epithelium & iris. The wing-cell layer was a barrier to gene-delivery to the more superficial epithelial cells. With the same experimental paradigm, but using square wave pulses of opposite polarity we noted significant gene expression in the iris but limited gene expression in the cornea restricted to the stroma close to the endothelial cell layer. Expression vectors injected into the anterior chamber without electrical pulses did not cause gene expression in the cornea or iris.
These results suggest that although electropermeabilization of the cell membrane was necessary for gene expression as evidenced by gene-expression in the cornea when constant-voltage pulses were used, both electrophoresis and passive tissue distribution contributed to the delivery of plasmids to targeted tissues. Topical application of plasmids to the cornea followed by electroporation did not result in measurable gene expression indicating that the route of delivery of the plasmids was important.
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