Aav-Mediated Dual-Axis Gene Therapy To Enhance Cartilage Repair

Abstract

The aim of this thesis project was to investigate the use of a recombinant adenoassociated virus (rAAV) as a gene therapy vector to target both the anabolic and catabolic axes of cartilage homeostasis in order to enhance cartilage repair capabilities and prevent joint degradation. The specific aims of the research were to 1) evaluate healing of full-thickness chondral defects in femoral trochlear ridges repaired with autologous chondrocytes transduced ex vivo with an rAAV5 vector overexpressing the anabolic protein, insulin-like growth factor I (IGF-I); 2) investigate the impact of post-transcriptional silencing of interleukin-1[beta] (IL-1[beta]) mediated by rAAV2 expressing a short hairpin IL-1[beta] silencing motif in chondrocytes cultured in an osteoarthritic model and; 3) elucidate the humoral and cellmediated immune response, and its impact on transgene expression, following direct intraarticular (IA) injection of rAAV2 and rAAV5 overexpressing IGF-I. Autologous chondrocytes transduced ex vivo with rAAV5-IGF-I were arthroscopically implanted, in a fibrin vehicle, into 15mm diameter full-thickness chondral defects created in the lateral trochlear ridges of horses. Transgene expression was assessed by serially quantifying IGF-I concentration in the synovial fluid. Defect healing was assessed arthroscopically at 8 weeks post-implantation. Long-term healing at 8 months post- implantation was assessed using gross, histological, immunohistochemical, and biochemical parameters. rAAV5-IGF-I treated defects had improved short and long-term healing with improved tissue architecture and collagen type II content compared to defects repaired with naïve chondrocytes. Transduction of chondrocytes with rAAV2 expressing a short hairpin IL-1[beta] led to significantly reduced IL-1[beta] mRNA expression following stimulation with lipopolysaccharide (LPS). Down-regulation of additional key catabolic cytokines and degradative enzymes, including tumor necrosis factor (TNF)-[alpha] and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5, was also seen in transduced cultures. Posttranscriptional silencing of IL-1[beta] appeared to limit the catabolic cascade seen in cartilage following LPS simulation. Direct intra-articular injection of rAAV2- and rAAV5-IGF-I led to prolonged transgene expression without any significant local joint inflammation. Carpal joints injected with rAAV5-IGF-I had significantly higher levels of IGF-I in the synovial fluid compared to rAAV2-IGF-I despite a more robust and diverse humoral immune response to AAV5. A cellmediated immune response was not noted in either treatment group.