title: Single intra-articular injection of adeno-associated virus results in stable and controllable in vivo transgene expression in normal rat knees.
abstract: OBJECTIVE
  To test the hypothesis that in vivo transgene expression mediated by single intra-articular injection of adeno-associated virus serotype 2 (AAV2) persists within intra-articular tissues 1 year post-injection and can be externally controlled using an AAV2-based tetracycline-inducible gene regulation system containing the tetracycline response element (TRE) promoter.
  
  
  METHODS
  Sprague Dawley rats received intra-articular injections of AAV2-cytomegalovirus (CMV)-enhanced green fluorescent protein (GFP) and AAV2-CMV-luciferase (Luc) into their right and left knees, respectively. Luciferase expression was evaluated over 1 year using bioluminescence imaging. After sacrifice, tissues were analyzed for GFP+ cells by fluorescent microscopy. To study external control of intra-articular AAV-transgene expression, another set of rats was co-injected with AAV2-TRE-Luc and AAV2-CMV-reverse-tetracycline-controlled transactivator (rtTA) into the right knees, and AAV2-CMV-Luc and AAV2-CMV-rtTA into the left knees. Rats received oral doxycycline (Dox), an analog of tetracycline, for 7 days. Luciferase expression was assessed by bioluminescence imaging.
  
  
  RESULTS
  Luciferase expression was localized to the injected joint and persisted throughout the 1-year study period. Abundant GFP+ cells were observed within intra-articular soft tissues. Transgene expression in AAV2-TRE-Luc injected joints was upregulated by oral administration of Dox, and downregulated following its removal, at 14 days and 13 months post-AAV injection.
  
  
  CONCLUSIONS
  This longitudinal in vivo study shows that sustained and stable AAV-mediated intra-articular transgene expression can be achieved through a single intra-articular injection and can be controlled using a tetracycline-controlled inducible AAV system in a normal rat knee model. Highly regulatable long-term intra-articular transgene expression is of potential clinical utility for development of treatment strategies for chronic intra-articular disease processes such as inflammatory and degenerative arthritis.
authors.affiliation: Cartilage Restoration Center, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
authors.forename: K A
authors.initials: KA
authors.lastname: Payne
authors.forename: H H
authors.initials: HH
authors.lastname: Lee
authors.forename: A M
authors.initials: AM
authors.lastname: Haleem
authors.forename: C
authors.initials: C
authors.lastname: Martins
authors.forename: Z
authors.initials: Z
authors.lastname: Yuan
authors.forename: C
authors.initials: C
authors.lastname: Qiao
authors.forename: X
authors.initials: X
authors.lastname: Xiao
authors.forename: C R
authors.initials: CR
authors.lastname: Chu
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism; D000229:Dependovirus / Q000378:metabolism*; D004195:Disease Models, Animal; D004318:Doxycycline / Q000008:administration & dosage / Q000494:pharmacology*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006614:Hindlimb / Q000378:metabolism*; D007270:Injections, Intra-Articular; D008137:Longitudinal Studies; D008156:Luciferases / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D017207:Rats, Sprague-Dawley; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000494:pharmacology; D019076:Transgenes / Q000187:drug effects*
chemical_list: D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D008156:Luciferases; D013752:Tetracycline; D004318:Doxycycline