Gene transfer to rabbit retina with electron avalanche transfection.
OBJECTIVE
Nonviral gene therapy represents a promising treatment for retinal diseases, given clinically acceptable methods for efficient gene transfer. Electroporation is widely used for transfection, but causes significant collateral damage and a high rate of cell death, especially in applications in situ. This study was conducted in the interest of developing efficient and less toxic forms of gene transfer for the eye.


METHODS
A novel method for nonviral DNA transfer, called electron avalanche transfection, was used that involves microsecond electric plasma-mediated discharges applied via microelectrode array. This transfection method, which produces synchronized pulses of mechanical stress and high electric field, was first applied to chorioallantoic membrane as a model system and then to rabbit RPE in vivo. Gene transfer was measured by using luciferase bioluminescence and in vivo fluorescent fundus photography. Safety was evaluated by performing electroretinograms and histology.


RESULTS
In chorioallantoic membrane, electron avalanche transfection was approximately 10,000-fold more efficient and produced less tissue damage than conventional electroporation. Also demonstrated was efficient plasmid DNA transfer to the rabbit retina after subretinal DNA injection and transscleral electron avalanche transfection. Electroretinograms and histology showed no evidence of damage from the procedure.


CONCLUSIONS
Electron avalanche transfection is a powerful new technology for safe DNA delivery that has great promise as a nonviral system of gene transfer.
Chalberg
Thomas W
TW
Departments of Genetics, Stanford University, Stanford, CA 94305, USA .
Vankov
Alexander
A
Molnar
Fanni E
FE
Butterwick
Alexander F
AF
Huie
Philip
P
Calos
Michele P
MP
Palanker
Daniel V
DV
D000818:Animals; D049033:Chorioallantoic Membrane / Q000378:metabolism; D004553:Electric Conductivity; D018274:Electroporation / Q000379:methods*; D004596:Electroretinography; D015870:Gene Expression / Q000502:physiology*; D008156:Luciferases / Q000235:genetics*; D008839:Microelectrodes; D008856:Microscopy, Fluorescence; D010957:Plasmids; D011817:Rabbits; D012160:Retina / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D008156:Luciferases