title: An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney.
abstract: BACKGROUND
  Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non-viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression.
  
  
  METHODS
  In this study, we examined the potential of ultrasound for gene transfer into the kidney. First, we transfected rat left kidney with luciferase plasmid mixed with microbubbles, Optison, to optimize the conditions (duration of ultrasound and concentration of Optison). Then, 4, 7, 14 and 21 days after gene transfer, luciferase activity was measured. Next, localization of gene expression was assessed by measuring luciferase activity and green fluorescent protein (GFP) expression. Expression of GFP plasmid was examined under a fluorescence microscope at 4 and 14 days after gene transfer. Finally, to examine the side effects of this gene transfer method, biochemical assays for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cre) were performed.
  
  
  RESULTS
  Optison and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 70-80% of total glomeruli could be transfected. Also, a significant dose-dependent effect of Optison was observed as assessed by luciferase assay (Optison 25%: 12.5 x 10(5) relative light units (RLU)/g tissue; 50%: 31.3 x 10(5) RLU/g tissue; 100%: 57.9 x 10(5) RLU/g tissue). GFP expression could be observed in glomeruli, tubules and interstitial area. Results of blood tests did not change significantly after gene transfer.
  
  
  CONCLUSIONS
  Overall, an ultrasound-mediated gene transfer method with Optison enhanced the efficiency of gene transfer and expression in the rat kidney. This novel non-viral method may be useful for gene therapy for renal disease.
authors.affiliation: Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, Suita 565-0871, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Koike
authors.forename: Naruya
authors.initials: N
authors.lastname: Tomita
authors.forename: Haruhito
authors.initials: H
authors.lastname: Azuma
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Taniyama
authors.forename: Keita
authors.initials: K
authors.lastname: Yamasaki
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Kunugiza
authors.forename: Katsuro
authors.initials: K
authors.lastname: Tachibana
authors.forename: Toshio
authors.initials: T
authors.lastname: Ogihara
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Morishita
mesh_terms: D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D001219:Aspartate Aminotransferases / Q000097:blood; D001806:Blood Urea Nitrogen; D003404:Creatinine / Q000097:blood; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D007678:Kidney Glomerulus / Q000166:cytology / Q000378:metabolism; D008081:Liposomes; D008156:Luciferases / Q000378:metabolism; D008297:Male; D045423:Microbubbles; D051381:Rats; D017208:Rats, Wistar; D018108:Respirovirus / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014465:Ultrasonics
chemical_list: D008081:Liposomes; D049452:Green Fluorescent Proteins; D003404:Creatinine; D008156:Luciferases; D001219:Aspartate Aminotransferases; D000410:Alanine Transaminase