Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

Abstract

Objective. Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage.Methods. We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems.Results. Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage.Conclusion. These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.Osteoarthritis (OA) is a progressive disease that affects diarthrodial joints and is mainly characterized by a gradual deterioration of the articular cartilage. A disturbed balance in cartilage metabolism is thought to play an important role in the pathogenesis of OA and to be a key factor in determining its progression. Over the course of OA, the cartilage loses major components of its extracellular matrix (ECM), such as proteoglycans and type II collagen (1). Proinflammatory cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor ␣, produced locally by the inflamed synovium likely contribute to the pathophysiology of OA (2). Articular chondrocytes are the focus of OA because of pathologic changes in their gene expression pattern (3), the loss of their capacity to synthesize cartilage-specific matrix molecules, and their increased production of matrixdegrading enzymes (4).Despite various therapeutic options, including systemic nonsteroidal antiinflammatory drugs, local corticosteroids, physical therapy, regular exercise, use of orthopedic appliances, or with the advent of disease-and structure-modifying drugs, the management of OA remains an unresolved problem, especially for patients who are too young to undergo endoprosthetic total joint replacement. The difficulty in treating OA is largely due to its slow and irreversible progression and to the limited intrinsic ability of the cartilage to reequilibrate its natural components. Application of therapeutic genes to OA cartilage may offer potent alternatives for reestab- Most of the current approaches to restoring the physiolog...

Benefits of Recombinant Adeno-Associated Virus (rAAV)-Mediated Insulinlike Growth Factor I (IGF-I) Overexpression for the Long-Term Reconstruction of Human Osteoarthritic Cartilage by Modulation of the IGF-I Axis

“…Even though application of these stimuli was capable of containing cartilage degradation, it was not sufficient to compensate for the loss of matrix elements and cells and to reestablish an original cartilage surface. Growth, transcription and enzymatic factors are potent candidates to achieve these goals because of their anabolic or mitogenic properties such as fibroblast growth factor 2 (FGF-2) (20,21), bone morphogenetic proteins 2 and 4 (BMP-2 and BMP-4) (22,23), transforming growth factor β (TGF-β) (22,24,25), transcription factor sex-determining region Y-type high mobility group box 9 (SOX9) (20,26,27), glucuronosyltransferase-I (28), bcl-2 (29) and telomerase (30). A critical agent that may have the strongest value to readjust the disturbed homeostasis in OA cartilage is insulinlike growth factor (IGF)-I, since it has the ability to influence concomitantly metabolic and proliferative processes, affording protection against extracellular matrix degradation in horse and rabbit articular cartilage explant cultures experimentally treated with proinflammatory cytokines (13,14,22).…”

“…In contrast with vectors derived from adenoviruses (6,10,13,14,(16)(17)(18)(19)21,22,25) and retroviruses (7,11,12,23,26,30) or with nonviral compounds (8,15,28,29), systems based on the replication-defective, nonpathogenic human adeno-associated virus (AAV) may provide better tools for OA, since recombinant AAV (rAAV) can deliver genes in nondividing cells such as chondrocytes both in vitro and in situ in their dense extracellular matrix at high efficiencies and for extended periods of time (20,24,27,31). Also, removal of the viral protein coding sequences in rAAV make them less immunogenic than adenoviral vectors characterized by shortterm transgene expression levels (32).…”

“…The study was approved by the Ethics Committee of the Saarland Physicians Council. Cartilage explant cultures (6.2-mm diameter, 2-mm thick) and articular chondrocytes were prepared as previously described (20,27,31,45). Human anterior cruciate ligament (ACL) was obtained in patients undergoing total knee arthroplasty (three patients, 70-76 years of age), and primary human ACL fibroblasts were prepared as previously described (46).…”

“…pAd8 contains the AAV-2 replication and encapsidation functions (48). rAAV-lacZ carries the lacZ gene for β-galactosidase (β-gal) under the control of the cytomegalovirus immediate-early (CMV-IE) promoter (20,27,31,45,49,50). rAAV-red fluorescent protein (rAAV-RFP) carries a 776-bp Discosoma sp.…”

“…rAAVs were packaged as conventional (not self-complementary) vectors in the 293 cell line, an adenovirus-transformed human embryonic kidney cell line, using adenovirus 5 and pAd8 for helper functions. The preparations were purified by dialysis and titered by real-time PCR (20,27,31,45,49,50), averaging 10 10 transgene copies/mL (ratio virus particles to functional vectors = 500/1) (49).…”