High-level gene transfer to the cornea using electroporation.
BACKGROUND
Methods for gene transfer to the cornea that yield high-level expression without inflammation or trauma are currently lacking. Because electroporation has proven effective for gene transfer in other tissues in terms of expression levels and safety, this study quantitatively evaluated its use in the cornea.


METHODS
To evaluate the use of electroporation in the mouse cornea, plasmids expressing either luciferase or green fluorescent protein were injected intracorneally or subconjunctivally and square-wave electric pulses were immediately applied to the eyes. Gene expression was quantified at later times and trauma and inflammation were monitored visually and by measuring interleukin-6 (IL-6) production.


RESULTS
The application of electric pulses to eyes injected with plasmid resulted in nanogram levels of gene product expression. At an optimal field strength of 200 V/cm, no trauma, corneal edema or inflammation was observed. However, at higher field strengths, corneal damage was detected. Compared with injection of DNA alone, up to 1000-fold more gene product was produced using electroporation. Expression was detected as early as 6 h post-electroporation, remained high for 3 days, and decreased by 7 days. Gene expression was detected over the entire surface of the cornea in both epithelial and stromal layers.


CONCLUSIONS
These results demonstrate that electroporation is an excellent method for delivering genes to multiple cell layers within the mouse cornea and that it results in extremely high levels of gene expression with little, if any, inflammatory response or tissue damage, making this a very useful technique for corneal gene transfer.
Blair-Parks
Kathleen
K
Department of Microbiology and Immunology, College of Medicine, University of South Alabama, Mobile, AL 36688, USA.
Weston
Bonnie C
BC
Dean
David A
DA
D000818:Animals; D003319:Corneal Stroma; D004247:DNA; D018274:Electroporation; D019573:Epithelium, Corneal; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D007267:Injections; D008081:Liposomes; D008156:Luciferases; D008164:Luminescent Proteins; D051379:Mice; D008807:Mice, Inbred BALB C
D008081:Liposomes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D008156:Luciferases