Research Fellow Mayo Clinic Rochester, Minnesota, United States
Background: The CMV promoter is widely considered a strong, constitutive promoter for driving transgene expression in the context of gene therapy. In the case of our therapeutic, which is starting a phase 1b clinical trial, the CMV promoter is used to drive expression of the interleukin-1 receptor antagonist (IL-1Ra) within joints for the treatment of arthritis, using AAV as the vector. However, in a recent equine study, 4-5 fold increased expression of IL-1Ra was seen in the joints of horses with arthritis compared to expression in normal joints. An inducible component to the CMV promoter is a potential explanation for this phenomenon. Because the intra-articular injection of AAV into equine joints transduces chondrocytes very efficiently, we evaluate this possibility in a series of in vitro experiments using human chondrocytes. Methods: Human chondrocytes were plated as monolayer in T-175 flasks. The following day, flasks underwent overnight transduction with AAV.GFP or AAV.IL-1Ra. Monolayers were then pelleted. Pellet media was collected at particular time points, and on day 14 IL-1b (1 ng/ml) was added to some wells. An additional media change and IL-1b washout period followed. On day 20, IL-1b was included in wells with previous exposure. Final media collection was day 23. Media was assayed for IL-1Ra expression by ELISA. Results: An increase in IL-1Ra was seen in chondrocytes exposed to IL-1b when looking at Day 17 and Day 23. The Day 17 IL-1b exposed and unexposed cells averaged 1503.2 pg/mL and 449.73 pg/mL IL-1Ra respectfully, p = 0.017; 95% CI [1192.08, 1814.32], 95% CI [90.27, 809.2]. Conclusion: Although CMV is seen as a constitutive promoter, these data suggest the presence of an inducible element, responsive to the inflammatory cytokine to IL-1b in human chondrocytes. Because arthritis is a disease in which the joint undergoes flares and remissions, this property would be useful in the setting of arthritis gene therapy. To better characterize this phenomenon, experiments currently being outlined include: seeing if TNF-a elicits a similar response, repeating this experiment in synovial fibroblasts, looking at how the presence of CREB and NF-kB inhibition affect results, and exploring how common OA therapies affect transduction and IL-1Ra production. Lastly, we recently got approval to bring this experiment in vivo with rabbits.
