Scientific Article   |    
Recombinant Adeno-Associated Virus-Mediated Osteoprotegerin Gene Therapy Inhibits Wear Debris-Induced Osteolysis
Michael Ulrich-Vinther, MD; Emily E. Carmody, BS; J. Jeffrey Goater, MA, MBA; Kjeld S�balle, MD; Regis J. O'Keefe, MD, PhD; Edward M. Schwarz, PhD
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Investigation performed at the Department of Orthopedics, Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, New York, and the Department of Orthopedics, �arhus University Hospital, �arhus, Denmark

Michael Ulrich-Vinther, MD
Emily E. Carmody, BS
J. Jeffrey Goater, MA, MBA
Regis J. O'Keefe, MD, PhD
Edward M. Schwarz, PhD
Department of Orthopedics, Center for Musculoskeletal Research (M.U.-V., E.E.C., J.J.G., and R.J.O'K.,) and Orthopaedic Research Laboratory (E.M.S.), University of Rochester Medical Center, 601 Elmwood Avenue, Box 665, Rochester, NY 14642. E-mail address for E.M. Schwarz: Edward_Schwarz@urmc.rochester.edu

Kjeld S�balle, MD
Department of Orthopedics, Aarhus University Hospital, DK-8000 �arhus C, Denmark

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases, Public Health Service Grants AR45971 and AR46545; and a research grant from the Orthopaedic Research and Education Foundation. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

J Bone Joint Surg Am, 2002 Aug 01;84(8):1405-1412
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Background: Aseptic loosening of orthopaedic implants secondary to wear debris-induced osteolysis is a serious problem. Osteoprotegerin (OPG) is a natural decoy protein that inhibits osteoclast activation and bone resorption. This study investigated whether gene therapy using a recombinant adeno-associated viral vector that expresses OPG can inhibit wear debris-induced osteolysis.

Methods: A recombinant adeno-associated virus (rAAV) vector co-expressing OPG (rAAV-OPG-IRES-EGFP) was generated. A control vector expressing b-galactosidase (rAAV-LacZ) was also prepared. In vitro validation experiments were performed to determine rAAV-OPG-IRES-EGFP transduction efficiency, OPG expression level and function in bone wafer, and osteoclastic activity.

The effect of rAAV-OPG-IRES-EGFP in vivo gene therapy on wear debris-induced osteolysis was then evaluated in a mouse calvarial model in which a single intramuscular injection of the vector was administered prior to the introduction of the wear debris. The effects of the rAAV-OPG-IRES-EGFP gene therapy on wear debris-induced osteoclastogenesis and bone resorption were determined by histomorphometry on day 10.

Results: In vitro experiments revealed that 100% of human embryonic kidney 293 cells were transduced at a multiplicity of infection of 1000 with both rAAV-OPG-IRES-EGFP and rAAV-LacZ. At a rAAV-OPG-IRES-EGFP multiplicity of infection of 1000, an OPG concentration of 135 ng/mL of culture media was achieved after four days. Using a bone-wafer assay for osteoclast activity, we found that treatment with rAAV-OPG-IRES-EGFP reduced resorption sevenfold compared with parathyroid hormone-stimulated controls and elevenfold compared with rAAV-LacZ controls. Furthermore, a seventeenfold decrease in RANKL and macrophage colony-stimulating factor-induced splenocyte osteoclastogenesis was observed in co-cultures containing rAAV-OPG-IRES-EGFP-infected fibroblasts.

In vivo administration of rAAV-OPG-IRES-EGFP resulted in detectable transduction of myocytes at the injection site and a significant increase in expression of serum OPG levels by the second day (p < 0.05). Maximal concentrations were obtained on day 6 and then leveled off throughout the observation period. In contrast, serum OPG could not be detected in the sham-treated, uninfected titanium-stimulated, or rAAV-LacZ-infected mice. In the control mice, titanium implantation resulted in a threefold increase in the mean number of osteoclasts adjacent to the sagittal suture as well as a twofold increase in the mean area of soft tissue in the sagittal suture compared with the sham-treated mice. In contrast, osteoclast numbers remained at basal levels, and the area of soft tissue in the sagittal suture was markedly reduced in titanium-implanted animals that received rAAV-OPG-IRES-EGFP treatment, demonstrating a complete inhibition of osteolysis in response to titanium particles.

Conclusions: A single intramuscular injection of the rAAV-OPG-IRES-EGFP vector can efficiently transduce myocytes to produce high levels of OPG. The OPG effectively inhibits wear debris-induced osteoclastogenesis and osteolysis.

Clinical Relevance: Currently, there is no approved drug therapy to prevent or inhibit periprosthetic osteolysis. Although preclinical studies have identified potential drug therapies (i.e., bisphosphonates), there is no evidence that these drugs can effectively treat aseptic loosening in patients. This is the first evidence that in vivo OPG gene therapy can be used to prevent wear debris-induced osteolysis.

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    These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.
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