Gene delivery with optimized electroporation parameters shows potential for treatment of gliomas.
Electroporation, a standard laboratory method of introducing exogenous molecules into cells, has been gaining importance as a very effective non-viral physical technique of gene delivery. In this study, we have used subcutaneous model of the C6 rat glioma cells and established an optimal condition to obtain very high gene expression in tumor tissues using both reporter and functional genes. Tumors grown on the flanks of Wistar rats are exposed and directly injected with plasmid DNA having the constructs of luciferase, green fluorescent protein and, the fragment of the diphtheria toxin, DT-A. The tumors are then subjected to square wave pulses from an electroporator. Gene expression is found to be several orders of magnitude higher when the tumors are pulsed with the optimized electrical parameters compared to the controls. For luciferase, the enhancement is approximately 135-fold, for the green fluorescent protein, gene expression is seen over a wide area within the sections examined, as contrast to a few punctate dots in the control specimens, and finally, DT-A shows massive death in the tumor tissue. A special circular array of six needles through which pulses are delivered with rotating electric field is found to be highly efficient in transferring genes inside the tumor. Direct injection of plasmid DNA followed by electroporation allows very high in vivo gene transfer and its subsequent expression into tumor tissues. This method may be applicable to any solid tumor.
Yoshizato
K
K
Department of Neurosurgery, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan.
Nishi
T
T
Goto
T
T
Dev
S B
SB
Takeshima
H
H
Kino
T
T
Tada
K
K
Kimura
T
T
Shiraishi
S
S
Kochi
M
M
Kuratsu
J I
JI
Hofmann
G A
GA
Ushio
Y
Y
D000818:Animals; D001932:Brain Neoplasms / Q000628:therapy*; D004167:Diphtheria Toxin / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D015316:Genetic Therapy; D005910:Glioma / Q000628:therapy*; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D010957:Plasmids; D051381:Rats; D017208:Rats, Wistar
D004167:Diphtheria Toxin; D005819:Genetic Markers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases