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

骨关节炎（OA）是最常见的关节炎形式，在英国影响近900万人，但没有有效的治疗方法。因此，对于减缓疾病进展和改善患者生活质量的新的和更好的疗法存在显著未满足的临床需求。几十年来，使用负责软骨破坏的酶的内源性抑制剂（如TIMP-3）的可能性一直是一种有趣的可能性。然而，尽管进行了多年的研究，但许多挑战阻碍了TIMP-3作为OA疗法的发展，包括不可接受的副作用和产生足够量的蛋白质的问题。我们现在已经克服了这些困难，通过工程TIMP-3选择性地靶向聚集蛋白聚糖酶，仅用于抑制和开发一种新的蛋白质生产方法，导致重组TIMP-3的生产增加10倍。本项目的目的是开发一种新的治疗策略，用于使用这种工程化的TIMP-3抑制聚集蛋白聚糖酶治疗OA。这里提出的策略是采用一种新的方法（生物技术）将这种分子传递到体内关节炎关节。这种方法使高浓度的局部治疗分子的疾病的网站（S），从而消除副作用和增加therapeutic efficacy.Aims and ObjectivesThe的目的，这一建议是开发一种新的治疗策略治疗OA。我们的假设是，局部递送工程化TIMP-3将通过获得高局部浓度的TIMP-3来改善受OA影响的关节中的软骨破坏，所述TIMP-3将抑制聚集蛋白聚糖酶活性。这些目标将通过完成以下目标来实现：1.使用GFP融合蛋白技术表达和纯化大量潜在的工程化TIMP-3。2.在小鼠疾病模型中确定潜在工程化TIMP-3对OA的发展和进展的影响.表征工程TIMP-3对参与OA发病机制的酶的ADAM和ADAMTS家族成员的抑制作用。潜在的应用和益处OA没有有效的治疗方法，目前的治疗旨在减轻OA患者的疼痛。然而，这些疗法对阻止疾病进展没有效果，因此目前终末期OA通过关节置换手术治疗。这项工作的应用可能会转化为一种新的OA治疗方法，也许与现有的治疗方法相结合，以创建一种治疗方案，减少关节炎关节中的软骨退化和随后的软骨下骨增厚。这可以显著减少关节损伤，从而减少疾病进展，从而为患者提供改善的生活质量，并可能延迟或避免关节置换手术。这里提出的方法有许多优点。首先，通过使用宿主免疫系统不识别的天然存在的蛋白质的重组形式，这种治疗的副作用的风险被最小化。其次，治疗分子在疾病部位的局部活化将导致高局部浓度，并进一步降低脱靶效应的风险。最后，我们设计了TIMP-3分子，以增加其特异性，从而避免了以前用合成酶抑制剂看到的副作用。此外，这种策略具有很大的翻译潜力。本提案中概述的研究可能会导致临床试验，以制定中期（7-10年）治疗OA的新策略。

Context of researchOsteoarthritis (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 ObjectivesThe 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 benefitsThere 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).