Gene transfer to corneal epithelium and keratocytes mediated by ultrasound with microbubbles.
OBJECTIVE
The cornea is an ideal organ for evaluating gene transfer because it can be treated noninvasively and monitored easily. The present study was performed to investigate the practical efficacy and safety of ultrasound (US) plus microbubble (MB)-mediated gene transfer to cornea.


METHODS
Cultured rabbit corneal epithelial (RC-1) cells were incubated in 24-well dishes with plasmid DNA having a green fluorescent protein (GFP) gene under a cytomegalovirus promoter. The cells were exposed to US under different intensities (1 MHz; power, 0.5 approximately 2 W/cm2; duration, 15-120 seconds; duty cycle, 20%-100%). The effect of simultaneous stimulation with MBs was also examined. Gene transfer was quantified by counting the number of GFP-positive cells under microscopy. Furthermore, in vivo gene transfer was examined by GFP plasmid injection into rabbit cornea and US exposure with MBs.


RESULTS
In the in vitro study, DNA exposure alone could not transfer gene into cultured RC-1 cells; US enhanced gene transfer slightly. Coexposure with MBs significantly increased gene transfer efficiency. In the in vivo study, DNA injection alone could transfer the gene to a limited degree, but plasmid injection plus US with MBs strongly increased gene transfer efficiency without apparent tissue damage, and gene transfer was achieved two dimensionally.


CONCLUSIONS
US with MBs greatly increases gene transfer to in vivo and in vitro corneal cells. This noninvasive gene transfer method may be a useful tool for clinical gene therapy.
Sonoda
Shozo
S
Department of Ophthalmology, Kagoshima University, Japan.
Tachibana
Katsuro
K
Uchino
Eisuke
E
Okubo
Akiko
A
Yamamoto
Matsuo
M
Sakoda
Kenji
K
Hisatomi
Toshio
T
Sonoda
Koh-Hei
KH
Negishi
Yoichi
Y
Izumi
Yuichi
Y
Takao
Sonshin
S
Sakamoto
Taiji
T
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D003287:Contrast Media / Q000378:metabolism; D003319:Corneal Stroma / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005466:Fluorocarbons / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D049408:Luminescent Agents / Q000378:metabolism; D008297:Male; D045423:Microbubbles; D010957:Plasmids / Q000235:genetics; D011817:Rabbits; D019076:Transgenes; D014465:Ultrasonics
D003287:Contrast Media; D005466:Fluorocarbons; D049408:Luminescent Agents; D049452:Green Fluorescent Proteins; C042852:perflutren