Regional Gene Therapy for Full-Thickness Articular Cartilage Lesions Using Naked DNA with a Collagen Matrix Paul E. Di Cesare, 1 Sally R. Frenkel, 1 Cathy S. Carlson, 2 Carrie Fang, 1 Chuanju Liu 1 1 Musculoskeletal Research Laboratory, New York University–Hospital for Joint Diseases, Department of Orthopaedic Surgery, 301 East 17th Street, Suite 1500, New York 10003 2 Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55126 Received 6 June 2005; accepted 30 December 2005 Published online 11 April 2006 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jor.20143 ABSTRACT: A novel gene therapy approach for treating damaged cartilage is proposed that involves placing endotoxin-free cDNA containing the gene for bone morphogenetic protein-2 (BMP-2) in type I collagen sponges and then transferring the naked plasmid DNA construct to the injury site. A full- thickness cartilaginous defect in rabbits implanted with plasmid containing a marker gene (b- galactosidase) showed expressed protein as detected by immunostaining. At 1 week postimplanta- tion, mesenchymal cells subjacent to the defect had incorporated the implanted naked plasmid DNA and, once transfected, served as local bioreactors, transiently producing the gene product. Plasmids containing the gene for BMP-2 implanted in collagen sponges in cartilage lesions stimulated hyalinelike articular cartilage repair at 12 weeks postimplantation, nearly equivalent in quality to that induced by collagen sponges with recombinant BMP-2 protein. Our approach circumvents the risks of inflammation and immunogenic response associated with the use of viral vectors. Naked plasmid DNA as a vehicle for transferring therapeutic genes has been shown to be effective in a therapeutic model within rabbit articular cartilage and appears to be safe and cost effective. ß 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 24:1118– 1127, 2006 Keywords: cartilage repair; gene therapy; naked DNA; bone morphogenetic protein-2 (BMP-2); collagen sponge INTRODUCTION Normal joint function requires a smooth articular surface composed of hyaline cartilage. The response of normal articular cartilage to injury or damage often results in suboptimal repair because of the limited regenerative and reparative capabilities of chondrocytes. 1,2 Many of the cur- rent treatment options (e.g., drilling, microfrac- ture, abrasion arthroplasty) are primarily aimed at stimulating extrinsic repair from cells deep to the tidemark. These techniques may temporarily alleviate clinical symptoms, but none has resulted in the regeneration of tissue with structural, biochemical, and biomechanical properties resem- bling those of normal hyaline cartilage. 3,4 Several peptide growth and differentiation fac- tors have been identified that appear to control cellular events associated with cartilage formation and repair, including transforming growth factor b (TGF-b), 5–7 fibroblastic growth factor, 8 insulinlike growth factor, 9 and bone morphogenetic proteins (BMPs). 10–14 BMPs are soluble extracellular pro- teins that can control osteogenic cell fate, initiate chondrogenesis, and maintain chondrocyte pheno- type. Fetal osteoblasts in vitro and in vivo normally express BMP genes, 15 and recombinant BMPs initiate cartilage and bone progenitor cell differ- entiation. 16,17 BMPs-1, -2, -4, -5, -6, and -11 and BMP receptors IA and II are expressed in a similar fashion in both fetal and adult cartilage, both normal and osteoarthritic; BMPs-7 and -8 and BMP receptor R-IB are expressed in fetal but not in adult cartilage. 18 BMP-2 is a potent stimulator of articular cartilage proteoglycan synthesis in vitro and in vivo. 19,20 BMP-3 and BMP-4 prevent chondrocyte dedifferentiation and maintain the differentiated phenotype in vitro. 21 Growth and differentiation factors have been used to facilitate articular cartilage repair. 22–24 These factors, which are expensive, may be degraded, diffuse 1118 JOURNAL OF ORTHOPAEDIC RESEARCH MAY 2006 Correspondence to: Paul E. Di Cesare (Telephone: 212-598- 6567; Fax: 212-598-6096; E-mail: pedicesare@aol.com) ß 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.