title: In vitro and in vivo testing of a novel regulatory system for gene therapy for intervertebral disc degeneration.
abstract: METHODS
  In vitro and in vivo testing of a gene expression control system.
  
  
  OBJECTIVE
  The purpose of this study is to establish the ability of controlling gene expression using an adeno-associated viral vector containing a novel control system (AAV-RheoSwitch GFP [Intrexon Corp., Blacksburg, VA]) in intervertebral disc cells for potential use in gene therapy trials.
  
  
  BACKGROUND
  Gene therapy for disc degeneration shows promise; however, concern remains regarding safety. Careful control of gene expression is needed to facilitate translation into clinical trials.
  
  
  METHODS
  Rabbit nucleus pulposus cells were treated in vitro with increasing multiplicities of infection of AAV-RheoSwitch GFP, followed by increasing concentrations of Intrexon's activator ligand, and examined for fluorescence during and after removal of ligand. New Zealand white rabbits were injected with AAV-RheoSwitch GFP and killed either before or after 5 days of daily ligand injection. Tissues were analyzed for the presence of green fluorescent protein (GFP) with fluorescence microscopy and immunohistochemical staining.
  
  
  RESULTS
  In vitro, GFP expression was noted to be dose and time dependent, decreased 24 hours after removal of ligand, and was minimally detectable in cells after 48 hours. In vivo, increasing GFP expression was seen in animals treated with viral vector and ligand. No GFP expression was evident in tissues from rabbits that received only virus, or activator ligand alone. In addition, no GFP expression was evident in the adjacent discs, spinal cord, dura, bone, liver, or brain of any animals.
  
  
  CONCLUSIONS
  These data demonstrate that in vitro ligand-induced gene expression can be stimulated and effectively turned off by removal of the ligand. In addition, we demonstrated the in vivo utility of this system through showing up-regulation of GFP without nonspecific gene expression or expression in adjacent tissues. This system, therefore, has the potential to increase the safety of gene therapy in the treatment of intervertebral disc degeneration.
authors.affiliation: Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA. sowaga@upmc.edu
authors.forename: Gwendolyn
authors.initials: G
authors.lastname: Sowa
authors.forename: Edward
authors.initials: E
authors.lastname: Westrick
authors.forename: Corey
authors.initials: C
authors.lastname: Pacek
authors.forename: Paulo
authors.initials: P
authors.lastname: Coelho
authors.forename: Devin
authors.initials: D
authors.lastname: Patel
authors.forename: Gianluca
authors.initials: G
authors.lastname: Vadala
authors.forename: Helga
authors.initials: H
authors.lastname: Georgescu
authors.forename: Nam
authors.initials: N
authors.lastname: Vo
authors.forename: Rebecca
authors.initials: R
authors.lastname: Studer
authors.forename: James
authors.initials: J
authors.lastname: Kang
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D008024:Ligands; D011817:Rabbits; D014162:Transfection; D015854:Up-Regulation
chemical_list: D008024:Ligands; D049452:Green Fluorescent Proteins