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In Vitro and in Vivo Testing of a Novel Regulatory System for Gene Therapy for Intervertebral Disc Degeneration

Sowa, Gwendolyn, MD, PhD*,‡; Westrick, Edward, MD*; Pacek, Corey, MD*; Coelho, Paulo, MS*; Patel, Devin*; Vadala, Gianluca, MD; Georgescu, Helga, BS*; Vo, Nam, PhD*; Studer, Rebecca, PhD*,§; Kang, James, MD*

doi: 10.1097/BRS.0b013e3181ed11c1
Basic Science

Study Design. 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.

Summary of Background Data. 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.

Conclusion. 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.

Gene therapy for intervertebral disc degeneration will require improved safety methods. We report the utility of a novel ecdysone receptor based control system, demonstrating in vitro dose and time dependent regulation through use of an activator ligand. In addition, in vivo utility is demonstrated without any toxicity or nonspecific expression.

*Ferguson Laboratory for Orthopaedic Research, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA

Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA

Department of Orthopaedic and Trauma Surgery, Campus Bio-Medico, University of Rome, Rome, Italy

§VAPHS 151-W University Dr C Pittsburgh, PA

Address correspondence and reprint requests to Gwendolyn Sowa, MD, PHD, Department of Orthopaedic Surgery, University of Pittsburgh, 3471 5th Ave, Ste 201, Pittsburgh, PA 15213; E-mail:

Acknowledgment date: October 22, 2009. Revised date: December 23, 2009. Accepted date: June 14, 2010.

The manuscript submitted does not contain information about medical device(s)/drug(s).

No funds were received in support of this work. No benefits in any form have been or will be received from a commercial party related directly or indirectly to the subject of this manuscript.

Viral vectors and ligand was generously provided through a material transfer agreement with Intrexon Corporation.

© 2011 Lippincott Williams & Wilkins, Inc.