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(Investigative Ophthalmology and Visual Science. 2006;47:558-564.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0889
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Gene Transfer to Corneal Epithelium and Keratocytes Mediated by Ultrasound with Microbubbles

Shozo Sonoda,1 Katsuro Tachibana,2 Eisuke Uchino,1 Akiko Okubo,1 Matsuo Yamamoto,3 Kenji Sakoda,4 Toshio Hisatomi,5 Koh-Hei Sonoda,5 Yoichi Negishi,6 Yuichi Izumi,4 Sonshin Takao,7 and Taiji Sakamoto1

1From the Departments of Ophthalmology and 4Periodontology and the 7Research Center for Life Science Resources, Kagoshima University, Kagoshima, Japan; the 2Department of Anatomy, Fukuoka University School of Medicine, Fukuoka, Japan; the 3Department of Periodontology, Showa University School of Dentistry, Tokyo, Japan; the 5Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; and the 6School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan.

PURPOSE. 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, 0.5~2 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.






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