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 α, 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 matrix-degrading 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 reestablishing the structural integrity of the damaged cartilage architecture.
Most of the current approaches to restoring the physiologic balance in injured articular cartilage are based on the delivery of factors that modulate the metabolic functions of chondrocytes, such as agents that counteract the processes of matrix degradation or ones that enhance the synthesis of matrix components. Protective effects of an IL-1 receptor antagonist (IL-1Ra) sequence against IL-1–induced cartilage breakdown have been documented in experimental models ex vivo (5, 6) and in vivo (7, 8). The transfer of the gene for a heat-shock protein (Hsp70) was also shown to afford protection against cellular injuries in chondrocytes (9). Alternatively, the delivery of sequences encoding for growth and enzymatic factors can potentially stimulate cartilage anabolism in vitro and in situ, such as insulin-like growth factor 1 (IGF-1) (10), fibroblast growth factor 2 (FGF-2) (11), bone morphogenetic protein 7 (BMP-7) (12), transforming growth factor β (TGFβ) (13), and glucuronosyltransferase I (14). However, application of external stimuli to damaged articular cartilage has not yet proved sufficient in fully reestablishing an original cartilage surface, and little is known about the effects on human OA cartilage. Other avenues of research may thus have value in the identification of supplementary treatments for OA.
Strategies to correct the altered gene expression patterns in OA chondrocytes may prove beneficial in readjusting the disturbed cartilage homeostasis. Transcription factors are key regulators of cartilage metabolism since they stimulate chondrogenesis in physiologic and pathologic conditions. Among them, SOX9, a member of the sex-determining region Y–type high mobility group box family of DNA binding proteins, plays critical roles in the regulation of skeletal and cartilage formation (15) and chondrocyte differentiation (16). SOX9 is expressed during embryonic development in a pattern that closely parallels that of cartilage matrix synthesis (17) and exerts its properties by activating the gene for type II collagen and other cartilage-specific genes (17–20). Notably, the levels of SOX9 expression decline in OA cartilage (21, 22). Modulation of the chondrocyte phenotype in OA cartilage by genetically modifying the levels of intracellular SOX9 expression might be advantageous in shifting the disrupted balance toward the synthesis of ECM components and contribute to the reproduction of an original articular cartilage surface.
A prerequisite for the development of an applicable gene treatment against OA is the ability of the gene vehicle to mediate the efficient and sustained expression of a candidate agent in order to counterbalance the progression of the disease. Recombinant adeno-associated virus (rAAV) vectors are particularly well suited for this purpose because they can transfer genes into human OA chondrocytes in vitro and in situ with high efficiencies and for extended periods of time (23–25). The rAAV vectors are derived from a replication-defective human parvovirus that is nonpathogenic in humans. The rAAV can transduce both dividing and nondividing cells, such as chondrocytes, and they drive transgene expression from highly stable episomes, which can persist for months to years (26). In addition, rAAV exhibit a reduced immunogenicity due to the complete removal of the viral protein coding sequences in the recombinant genome. These features are in marked contrast with the properties of other classes of vectors (27), such as retroviral vectors, which necessitate the division of the target cells (28), or adenoviral vectors, which generally mediate only short-term transgene expression (29).
In the present study, we tested the hypothesis that SOX9 overexpression via rAAV promotes the synthesis of proteoglycans and type II collagen in human OA chondrocytes in vitro and in cartilage explants in situ. We also evaluated whether application of the SOX9 vector restores the cartilage matrix in human OA cartilage as compared with normal cartilage.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Application of candidate therapeutic genes that restore the ECM in OA articular cartilage is an attractive approach to balancing the progression of the disease. In this regard, the effects of external stimuli provided by the expression of potent chondroprotective and chondroregenerative factors, such as IL-1Ra or IGF-1, have been widely evaluated (6, 10, 11), but complete reproduction of an original articular cartilage surface has not been achieved thus far with the use of these agents. Additional treatments might be required to expand the processes of regeneration in OA cartilage, such as those based on the use of factors that regulate chondrogenesis. Transcription factors are good candidates because they have the potential to correct the phenotype of OA chondrocytes and might thus contribute to the restoration of cartilage homeostasis.
The identification of a suitable gene vehicle is particularly important for the treatment of a slow, progressive disease such as OA, in which the effects of an agent may be required over prolonged periods of time. Vectors based on AAV might be advantageous to achieving this goal because they have the unique ability to mediate both efficient and stable transgene expression throughout the entire depth of cartilage (23, 24), in marked contrast with the properties of other classes of vectors (27). In the present study, we evaluated the ability of rAAV-mediated SOX9 overexpression to restore the cartilage matrix in human OA cartilage as compared with normal cartilage.
Our data indicate that sustained expression of a SOX9 gene cassette significantly increased the proteoglycan and type II collagen content in 3-dimensional cultures of human normal and OA chondrocytes, consistent with the effects of SOX9 upon the expression of cartilage matrix components (17–19, 43). Notably, the amount of proteoglycans and type II collagen noted in the treated OA chondrocytes was significantly higher than that in the control normal chondrocytes. Application of the rAAV-FLAG-hSOX9 vector did not promote cell proliferation in these systems, which is consistent with the properties of the transcription factor (26). These data demonstrate the ability of a therapeutic candidate to stably restore matrix synthesis in human OA chondrocytes. Delivery of a SOX9 gene cassette via adenoviral and retroviral vectors has been shown to enhance the type II collagen and glycosaminoglycan content in OA chondrocytes (43, 44), but the effects reported were incomplete in the absence of anabolic supplements and were noted only over a short period of time, in contrast with our findings using rAAV. This might be the result of the persistence of the rAAV transgenes in their targets (26), in contrast with adenoviral or retroviral transgenes that are either less stable or require cell division prior to expression.
Direct application of rAAV-FLAG-hSOX9 to human normal and OA cartilage in situ mediated high levels of transgene expression that was distributed throughout the thickness of the explant cultures, probably due to the ability of these small vectors to penetrate the dense ECM of the cartilage (23, 24). The manipulation of vectors such as rAAV might thus be desirable for introducing candidate genes directly into the targets as compared with other classes of vectors still used in ex vivo gene transfer protocols (5, 12, 45). While no effects on cell proliferation were seen, expression of the SOX9 gene cassette promoted a significant, dose-dependent increase in the proteoglycan and type II collagen content in OA cartilage to levels higher than or similar to those in the untreated normal cartilage and to depths relevant for clinical applications (24).
To the best of our knowledge, this is the first evidence of the ability of SOX9, or of any other therapeutic candidate, to compensate for the loss of ECM components in human OA cartilage. Interestingly, in the present system, matrix synthesis was enhanced in the treated OA cartilage as compared with the control normal cartilage. It remains to be seen whether altered (different from normal) levels of proteoglycans and type II collagen caused by SOX9 overexpression influence the structure of articular cartilage over time. Kypriotou et al (46) reported that concentrations of SOX9 that were too high might inhibit type II collagen expression in chondrocytes by disturbing the cellular balance between transcription factors, depending on the stage of cell differentiation. Regulation of SOX9 expression in these cells will be critical to the development of an appropriate gene treatment for OA that does not alter the integrity of the cartilage. Instead of using the strong CMV-IE transcription element, transgene expression might be controlled by regulatable (tetracycline-sensitive) or tissue-specific (SOX9, type II collagen, cartilage oligomeric matrix protein) promoters.
Kypriotou et al (46) also suggested that SOX9 alone might not be sufficient to reorient the chondrocyte phenotype. Accordingly, Ikeda et al (16) showed that codelivery of the genes for SOX5 and SOX6 with SOX9 (the SOX trio) was more efficient for inducing a chondrocyte phenotype in human mesenchymal stem cells in vitro as compared with SOX9 treatment alone, although effects of the SOX trio on OA cartilage have not yet been reported. Indeed, regeneration of an original cartilage surface was not afforded by administration of the SOX9 vector in the present study. Renewal of a native phenotype in OA chondrocytes might thus require the action of more than one intracellular therapeutic agent. This may be achieved by applying combinations of rAAV vectors at the same time (47). Restoration of a normal architecture in OA cartilage may certainly benefit from coapplication of factors that stimulate the metabolic and proliferative responses of the chondrocytes, such as IGF-1 (10, 48, 49), FGF-2 (11, 48), TGFβ (49), BMP-7 (12), IL-1Ra (5), and Hsp70 (9). Nevertheless, recovery from cartilage degradation using direct application of gene treatments will be practicable only if some cartilage surface and resident chondrocytes are maintained, as in the early stages of OA, while methods based on the transplantation of genetically modified cells or progenitors might be desirable for more advanced cases of the disease.
In summary, the results of this study indicate that direct, rAAV-mediated overexpression of SOX9 within human OA cartilage restores the synthesis of proteoglycans and type II collagen, 2 key ECM components of the cartilage. Additional studies in established experimental models (7, 8) will be required in order to evaluate the effects of SOX9 overexpression in OA cartilage in vivo. The present findings provide motivation to further develop this therapeutic gene-transfer approach to the treatment of OA in humans.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Dr. Cucchiarini had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Drs. Cucchiarini and Madry.
Acquisition of data. Dr. Cucchiarini, Ms Thurn, and Ms Weimer.
Analysis and interpretation of data. Dr. Cucchiarini, Ms Thurn, Ms Weimer, and Drs. Kohn, Terwilliger, and Madry.
Manuscript preparation. Drs. Cucchiarini, Terwilliger, and Madry.
Statistical analysis. Dr. Cucchiarini.