Development of Sustained Release, Anti-Cytokine Short Interfering RNA (siRNA) Therapy for Equine Osteoarthritis

Synovitis is a key mediator of osteoarthritis (OA) and in the perpetuation of cartilage degradation, and approximately 60% of lameness in the horse has previously been attributed to OA. Synovial inflammation is characterised by a mononuclear cell infiltration into the synovial membrane, with concurrent increases in catabolic cytokines predominantly produced by synovial macrophages. These cytokines cause changes in fibroblast-like synoviocyte gene expression, including the up-regulation of damaging cartilage matrix degrading proteinases and further cytokines. It was therefore hypothesised that the synovial membrane may represent a disease-modifying target. When referring to synovial macrophages and fibroblast-like synovioctyes collectively, the term synoviocytes is used. The general aims of this thesis were to design and test an anti-cytokine therapy; short interfering RNA (siRNA), and to analyse its potential with respect to decreasing target catabolic cytokines produced by synoviocytes. Delivery of this therapy to synoviocytes via biodegradable polymer microspheres was also examined. The effects of the therapy and therapydelivery system on synoviocyte and cartilage health, were assessed using in vitro co-culture systems, which were designed and used to elucidate interactions between the two predominant cell populations present during synovitis; synovial macrophages and fibroblast-like synoviocytes. Successful equine anti-IL-1ß siRNA was designed and tested on equine synovoicytes. These cells were also shown to successfully phagocytose 2- 6µm biodegradable polymer microspheres in a highly efficient manner. A novel multi-species in vitro model was created to quantify cell-specific responses to inflammatory stimulation, of both macrophages and fibroblastlike synoviocytes in co-culture. This model revealed that macrophages can stimulate fibroblast-like synoviocyte mitosis, and modulate its ADAMTS5 mRNA expression in an inflammatory environment. Using an inflammatory stimulated co-culture model involving a mixed culture of macrophages, fibroblast-like synoviocytes and cartilage explants, it was observed that the mixed synoviocyte culture can also act to moderate the inflammatory response. Synoviocytes were shown to exert a protective mechanism over cartilage glycosaminoglycan release, whilst driving the inflammatory response through the production of pro-inflammatory cytokines. This thesis has successfully provided the initial design and testing of an equine anti-IL1ß therapy and delivery vehicle in vitro, providing a platform for further experimentation to assess the efficiency of delivery and therapeutic potential when used together.