rAAV-Mediated <i>sox9</i> Overexpression Improves the Repair of Osteochondral Defects in a Clinically Relevant Large Animal Model Over Time In Vivo and Reduces Perifocal Osteoarthritic Changes.

Abstract

Gene transfer of the transcription factor SOX9 with clinically adapted recombinant adeno-associated virus (rAAV) vectors offers a powerful tool to durably enhance the repair process at sites of osteochondral injuries and counteract the development of perifocal osteoarthritis (OA) in the adjacent articular cartilage. To examine the ability of an rAAV <i>sox9</i> construct to improve the repair of focal osteochondral defects and oppose perifocal OA development over time in a large translational model relative to control gene transfer. Controlled laboratory study. Standardized osteochondral defects created in the knee joints of adult sheep were treated with rAAV-FLAG-h<i>sox9</i> relative to control (reporter) rAAV-<i>lacZ</i> gene transfer. Osteochondral repair and degenerative changes in the adjacent cartilage were monitored using macroscopic, histological, immunohistological, and biochemical evaluations after 6 months. The microarchitecture of the subchondral bone was assessed by micro-computed tomography. Effective, prolonged <i>sox9</i> overexpression via rAAV was significantly achieved in the defects after 6 months versus rAAV-<i>lacZ</i> treatment. The application of rAAV-FLAG-h<i>sox9</i> improved the individual parameters of defect filling, matrix staining, cellular morphology, defect architecture, surface architecture, subchondral bone, and tidemark as well as the overall score of cartilage repair in the defects compared with rAAV-<i>lacZ</i>. The overexpression of <i>sox9</i> led to higher levels of proteoglycan production, stronger type II collagen deposition, and reduced type I collagen immunoreactivity in the <i>sox9</i>- versus <i>lacZ</i>-treated defects, together with decreased cell densities and DNA content. rAAV-FLAG-h<i>sox9</i> enhanced semiquantitative histological subchondral bone repair, while the microstructure of the incompletely restored subchondral bone in the <i>sox9</i> defects was not different from that in the <i>lacZ</i> defects. The articular cartilage adjacent to the <i>sox9</i>-treated defects showed reduced histological signs of perifocal OA changes versus rAAV-<i>lacZ</i>. rAAV-mediated <i>sox9</i> gene transfer enhanced osteochondral repair in sheep after 6 months and reduced perifocal OA changes. These results underline the potential of rAAV-FLAG-h<i>sox9</i> as a therapeutic tool to treat cartilage defects and afford protection against OA. The delivery of therapeutic rAAV <i>sox9</i> to sites of focal injuries may offer a novel, convenient tool to enhance the repair of osteochondral defects involving both the articular cartilage and the underlying subchondral bone and provide a protective role by reducing the extent of perifocal OA.