title: Sustained transgene expression in intervertebral disc cells in vivo mediated by microbubble-enhanced ultrasound gene therapy.
abstract: METHODS
  In vivo studies using a rat model were performed to determine the feasibility of microbubble-enhanced ultrasound gene transfer technique to the intervertebral disc.
  
  
  OBJECTIVE
  1) To establish this microbubble-enhanced ultrasound gene therapy technique for intervertebral disc cells in vivo without using viral vectors and 2) to estimate the duration of transgene expression in vivo.
  
  
  BACKGROUND
  Intervertebral disc degeneration and associated spinal disorders remain a formidable problem. Although gene therapy approaches have been reported as having promising therapeutic potential to regenerate the disc, concerns over safety issues using recombinant viral vectors limits its application. Successful gene transfer using ultrasound has been reported in muscle and cardiovascular systems in vivo.
  
  
  METHODS
  Two different reporter plasmid DNA encoding green fluorescent protein (GFP) and firefly luciferase were used. Plasmid DNA was mixed with ultrasonography contrast agent (microbubbles) and injected into coccygeal intervertebral discs of Sprague-Dawley rats. The therapeutic ultrasound was irradiated on the surface of injected discs. Rats were killed 1, 3, 6, 12, and 24 weeks after gene transduction. Harvested nucleus pulposus tissues were used for evaluation of transgene expression. The intact discs were used as a control.
  
  
  RESULTS
  Seven days after gene transfection, considerable numbers of GFP-positive cells were observed in nucleus pulposus from the GFP-transfected group. Luciferase assay revealed that the ultrasound group demonstrated approximately an 11-fold increase in luciferase activity over the plasmid DNA-only group. Furthermore, transgene expression mediated by this method was observed, at least up to 24 weeks.
  
  
  CONCLUSIONS
  Our study indicated that ultrasound transfection method with microbubbles significantly enhanced transfection efficiency of plasmid DNA into the nucleus pulposus cells in vivo. Furthermore, the sustained transgene expression in vivo was possible up to 24 weeks. The long-term gene expression mediated by simple and safe procedure has important clinical applications, including the treatment of chronic types of disease such as degenerative disc diseases.
authors.affiliation: Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan. kotaro@med.kobe-u.ac.jp
authors.forename: Kotaro
authors.initials: K
authors.lastname: Nishida
authors.forename: Minoru
authors.initials: M
authors.lastname: Doita
authors.forename: Toru
authors.initials: T
authors.lastname: Takada
authors.forename: Ken-Ichiro
authors.initials: K
authors.lastname: Kakutani
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Miyamoto
authors.forename: Takatoshi
authors.initials: T
authors.lastname: Shimomura
authors.forename: Koichiro
authors.initials: K
authors.lastname: Maeno
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kurosaka
mesh_terms: D000818:Animals; D003050:Coccyx; D004247:DNA / Q000008:administration & dosage; D005240:Feasibility Studies; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007270:Injections, Intra-Articular; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism*; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D049408:Luminescent Agents; D008297:Male; D045423:Microbubbles; D010957:Plasmids / Q000008:administration & dosage; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014162:Transfection / Q000379:methods; D019076:Transgenes; D014465:Ultrasonics
chemical_list: D049408:Luminescent Agents; D049452:Green Fluorescent Proteins; D004247:DNA; D049409:Luciferases, Firefly