246. Intra-Articular Gene Transfer of miR-15b Attenuates Molecular Mediators of Hemophilic Arthropathy in a Murine Model of Hemophilia

Abstract

Hemophilic arthropathy is a major co-morbidity in patients with hemophilia and caused by recurrent bleeds (hemarthrosis) in the joints. Despite the availability of protein replacement therapy, newer and targeted strategies are required to prevent the onset of blood-induced arthropathy, particularly in patients who develop inhibitors. Thus, a thorough understanding of the molecular mediators contributing to joint damage becomes pivotal to not only pinpoint mechanisms but also reveal biomarkers of the disease and targets for further intervention. Recently, in a murine model of hemophilia A, we identified that nuclear factor (NF)-kB and its associated factors-hypoxia inducing factor (HIF)-1/2α, vascular epithelial growth factor (VEGF)-α and matrix metallo proteinases(MMP) 3, contribute to the development of arthropathy [J Thromb Haemost 2013; 11:293-306]. In addition, several microRNA (miR) were also differentially expressed following an episodic bleeding. One of these, miR-15b is known to modulate expression of hypoxic and pro-angiogeneic genes. To document the expression pattern of miR-15b during development of arthropathy, the acute hemarthrosis model of hemophhilia A was studied on 1,3,7 and 24h after a single-injury and the chronic hemarthrosis model was studied at 60, 75 and 90 days after multiple injury. We then measured miR-15b levels by qPCR at each one of these time points. miR-15b was downregulated (~1 to 4 fold) from 3hrs after a single bleeding episode to the 90-day time point where the animals have 2 to 6 bleedings into the joint cavity. The maximal down regulation (-4 fold) of miR-15b was observed at 60 days. These data suggest that miR-15b is consistently and progressively repressed from the onset of synovitis to the development of arthropathy in the murine model of hemophilia A. To test if intra-articular overexpression of this miR modulates the molecular biomarkers of joint disease, we administered an adeno-associated virus (AAV)5 vector encoding miR15b into the joints of a chronic model of hemarthrosis. Our results demonstrate that miR15b over-expression downregulates markers such as VEGF-α, HIF 2α (~70% and ~34% respectively) in affected joints. In addition, co-administration of AAV5-miR15b intra-articularly and AAV2-factor VIII intravenously, attenuated the expression of MMPs 1, 3, 9 and 14 (~14% to 60%). Sixty days after intra-articular gene therapy, there was a considerable decrease in the joint scores of animals treated intra-articularly with AAV5-miR-15b when compared to the control AAV5-luc vector (1.6 Vs 2.6). These data provide the first evidence that targeted and local AAV mediated gene delivery of miRs can be potentially used as therapeutic agent to ameliorate hemophilic arthropathy.