Targeted Cleavage of Matrix Molecules in a Neocartilage

描述(由申请人提供)：这个项目的目标是开发一种方法和模型，在这种方法和模型中，从软骨细胞(新关节)生长的组织中选择的细胞外基质蛋白可以在特定的位置被切割。将在细胞外基质分子、软骨细胞表达的转基因分子和产生的新的片段的遗传序列中插入翻译后的凝血酶蛋白水解酶识别位点。在组织发育的任何阶段加入凝血酶都可以切割这种转基因蛋白质。直接的应用是通过比较分子裂解前后新结构分子的机械性能来评估候选结构分子，即承载负荷的分子。如果候选分子是结构分子，则该分子裂解后的力学性能应该降低。开发该方法的动机是了解骨关节炎的机械破坏过程，其中细胞外基质分子被裂解酶消化，随后组织在相对正常的载荷下失败。目前扰乱疾病过程的策略包括寻找阻断该过程中关键分子的试剂。这种方法的一个重大挑战是识别要阻断的关键分子。识别结构分子的传统方法是用外源酶消化候选分子，并测量前后的机械性能。这种方法的问题是，可用的酶很少是单分子特异性的。建议的方法将允许特定的选定分子的消化(切割)，极大地扩大了可被评估为候选结构分子的基质分子的数量。该方法可能在组织工程和药物释放方面有其他应用。该方法将是了解骨性关节炎疾病过程的一个有价值的工具，并作为治疗干预的指南。 公共卫生相关性：骨关节炎(OA)是导致残疾的最大原因，但对疾病过程的机制，特别是软骨是如何退化的，了解很少。这个项目将开发方法来确定负载是如何通过软骨传递的，以及哪些特定的负载分子可能在骨关节炎过程中受到损害。对这些分子的识别将有助于开发能够延缓损害并为OA提供疾病修正治疗的药物。

DESCRIPTION (provided by applicant): The goal of this project is to develop a method and model in which a chosen extracellular matrix protein of a tissue grown from cartilage cells (neocartilage) can be cleaved at a specific site. Addition of a translated thrombin protease recognition site will be inserted into the genetic sequence of an extracellular matrix molecule, the genetically modified molecule expressed by chondrocytes, and a neocartilage generated. The genetically modified protein could be cleaved by the addition of thrombin at any point in the development of the tissue. The immediate application is to allow evaluation of candidate structural molecules, molecules that carry load, by comparison of the mechanical properties of the neocartilage before and after cleaving the molecule. If the candidate molecule is a structural molecule, the mechanical properties should be reduced after cleavage of that molecule. The motivation for developing the method is to understand the mechanical failure process in osteoarthritis, where extracellular matrix molecules are digested by lytic enzymes and subsequently the tissue fails under relatively normal loads. Current strategies for disrupting the disease process involve search for agents to block critical molecules in the process. A significant challenge in this approach has been identifying the critical molecules to block. The traditional approach to identifying structural molecules is by digesting candidate molecules with an exogenous enzyme and measuring mechanical properties before and after. The problem with this approach is that there are very few enzymes available that are single molecule specific. The proposed method would allow digestion (cleavage) of a chosen molecule with specificity, greatly enlarging the number of matrix molecules that could be evaluated as candidate structural molecules. The method may have other applications in tissue engineering and drug release. The approach would be a valuable tool in understanding the OA disease process and as a guide for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is the largest cause of disability, but there is poor understanding of the mechanisms of the disease process, particularly how cartilage is degraded. This project will develop methods to identify how load is transmitted through cartilage, and which specific load carrying molecules might be damaged during the OA process. Identification of these molecules would allow development of drugs that could retard the damage and provide a disease modifying treatment to OA.