Treatment of osteoarthritis by inhibition of aggrecanases using targeted delivery of engineered TIMP-3

Context of research
Osteoarthritis (OA) is the most common form of arthritis affecting nearly 9 million people in the UK, yet there are no effective treatments. As such, there is a significant unmet clinical need for new and better therapies to slow progression of disease and improve patient quality of life. The possibility of using endogenous inhibitors of the enzymes responsible for cartilage destruction, such as TIMP-3, has been an intriguing possibility for decades. However, despite many years of research a number of challenges have prevented development of TIMP-3 as a therapy for OA, including unacceptable side-effects and problems in producing sufficient quantities of protein. We have now overcome these difficulties by engineering TIMP-3 to selectively target aggrecanases only for inhibition and developing a novel method of protein production that results in a 10-fold increase in production of recombinant TIMP-3. The aim of this project is to develop a new therapeutic strategy for the treatment of OA using this engineered TIMP-3 to inhibit aggrecanases. The strategy proposed here is to employ a novel method (the LAP technology) of delivering this molecule to arthritic joints in vivo. This approach enables the delivery of high local concentrations of therapeutic molecules at site(s) of disease, thus eliminating side-effects and increasing therapeutic efficacy.

Aims and Objectives
The aim of this proposal is to develop a new therapeutic strategy for treatment of OA. Our hypothesis is that local delivery of engineered TIMP-3 will ameliorate cartilage destruction in joints affected by OA by achieving high local concentrations of TIMP-3 that will inhibit aggrecanase activity.
These aims will be achieved by the completion of the following objectives:
1. Express and purify substantial quantities of latent engineered TIMP-3 using the LAP fusion protein technology.
2. Determine the effects of latent engineered TIMP-3 on the development and progression of OA in a mouse model of disease.
3. Characterise the inhibitory effects of engineered TIMP-3 on members of the ADAM and ADAMTS families of enzymes involved in the pathogenesis of OA.

Potential applications and benefits
There are no effective treatments for OA and current therapies aim to alleviate the pain experienced by people with OA. However, these therapies have no effect on halting disease progression with the result that end-stage OA is currently treated by joint-replacement surgery. The applications of this work may translate into a new therapy for OA, perhaps in combination with existing therapies, to create a treatment regimen that reduces cartilage degradation and subsequent subchondral bone thickening in arthritic joints. This could significantly reduce joint damage so that disease progression is curtailed, thus providing patients with improved quality of life and possibly delaying or avoiding joint replacement surgery. There are a number of advantages to the approach proposed here. Firstly, the risks of side effects of this treatment are minimized by using a recombinant form of a naturally occurring protein that is not recognised by the host immune system. Secondly, local activation of therapeutic molecules at the site(s) of disease will result in high local concentrations and further minimise the risks of off-target effects. Finally, we have engineered the TIMP-3 molecule to increase its specificity thus avoiding the side-effects previously seen with synthetic enzyme inhibitors. Furthermore, this strategy has a great deal of translational potential. The research outlined in this proposal could lead to clinical trials for the development of a new strategy for treating OA in the medium term (7-10 years).

Joints affected by osteoarthritis (OA) have increased levels of matrix-degrading enzymes such as matrix metalloproteinases (MMP) and aggrecanases that can degrade the articular cartilage and result in joint damage and pain. TIMP-3 is a potent endogenous inhibitor of MMPs and aggrecanases. However, attempts to harness the therapeutic potential of TIMP-3 have been prevented by technical challenges associated with protein production and by observations that synthetic broad-spectrum inhibitors of MMPs caused unwanted side-effects. In this project, TIMP-3 molecules that have been engineered to narrow their inhibitory activity to aggrecanases but not MMPs, will be produced and delivered to sites of arthritic disease. Targeting of these proteins to arthritic joints will be achieved by producing TIMP-3 in fusion with the latency-associated peptide (LAP) of TGF-beta1. This renders the protein biologically inactive until it reaches the site of disease where TIMP-3 is released from the LAP by MMPs present in arthritic joints. The effect of delivery of engineered TIMP-3 on the course of disease in vivo will be assessed by measuring cartilage degradation and subchondral bone thickening by a combination of histology, immunohistochemistry and micro computed tomography. The inhibitory effects of engineered TIMP-3 on various members of the ADAM and ADAMTS families of matrix-degrading enzymes will also be assessed in vitro to provide insight into the potential benefits of this therapeutic strategy. Knowledge of the targeting potential of this approach in OA and of the effects of inhibition of aggrecanases in joints affected by OA could pave the way for a novel treatment for OA that could be used alone or in combination with existing therapies.

The immediate benefit of the proposed study will be the advancement of knowledge that will be shared with the wider academic scientific community. The knowledge generated will be of direct benefit to researchers with an interest in matrix biology, drug delivery and osteoarthritis (OA). The project will reveal insights into the therapeutic potential of TIMP-3 in vivo and into the role(s) of aggrecanases in driving the disease process at various stages of OA. The relatively novel method of delivery of an enzyme inhibitor to arthritic joints will also be of great interest to the pharmaceutical industry, one of whom (Stealthyx Therapeutics, Ltd) is already developing various latent molecules for testing in a number of disease contexts. The data generated here will be the first to describe an enzyme inhibitor, rather than a cytokine, delivered using this method. In addition, there will also be great personal benefit to the postdoctoral research fellow through training to become an independent researcher and developing skills and knowledge in a number of important techniques including a surgical mouse model of OA, analysis of micro-computed tomography data and preparation and analyses of tissues for histology and immunohistochemistry.

As this project has a high translational value, the long term benefit of the research will ultimately be to patients, clinicians and the NHS. This project will provide detailed pre-clinical knowledge of an alternative therapeutic strategy for the treatment of osteoarthritis, together with a novel method for delivery of therapeutic molecules. This work could provide a platform upon which to develop more targeted and effective therapies for OA that could improve the quality of patients' lives and represent significant savings to the NHS in the future.