title: Gene transfer to corneal epithelium and keratocytes mediated by ultrasound with microbubbles.
abstract: 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.
authors.affiliation: Department of Ophthalmology, Kagoshima University, Japan.
authors.forename: Shozo
authors.initials: S
authors.lastname: Sonoda
authors.forename: Katsuro
authors.initials: K
authors.lastname: Tachibana
authors.forename: Eisuke
authors.initials: E
authors.lastname: Uchino
authors.forename: Akiko
authors.initials: A
authors.lastname: Okubo
authors.forename: Matsuo
authors.initials: M
authors.lastname: Yamamoto
authors.forename: Kenji
authors.initials: K
authors.lastname: Sakoda
authors.forename: Toshio
authors.initials: T
authors.lastname: Hisatomi
authors.forename: Koh-Hei
authors.initials: KH
authors.lastname: Sonoda
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Negishi
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Izumi
authors.forename: Sonshin
authors.initials: S
authors.lastname: Takao
authors.forename: Taiji
authors.initials: T
authors.lastname: Sakamoto
mesh_terms: 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
chemical_list: D003287:Contrast Media; D005466:Fluorocarbons; D049408:Luminescent Agents; D049452:Green Fluorescent Proteins; C042852:perflutren