Sustained Biomaterial-mediated Inhibition of R-spondin 2 to Target Pathological Wnt Signaling in Post-Traumatic Osteoarthritis

摘要 调查人员：特里斯坦·梅尔兹博士(PI)是一名ESI生物医学工程师，专注于开发新的治疗方法- 创伤后骨关节炎(PTOA)的治疗。共同调查者库尔特·汉肯森、DVM博士和克雷格 杜瓦尔博士分别在Wnt信号和生物材料介导的组织再生方面拥有专业知识。 研究背景：规范的Wnt/β-catenin(CWNT)信号的过度激活越来越被认识到 作为PTOA关节退行性变的驱动因素。与PTOA相关的一种新的cWnt激动剂是R-Respondin 2 (Rsp2)，一些文献和我们的数据支持它是一种通过cWnt介导的诱导致病的配体。 滑膜纤维化、蛋白水解酶过度表达、软骨细胞肥大和异常骨硬化症的治疗。一个 最近的药物筛选发现，药物Mianserin通过阻断Lgr5结合来抑制Rsp2信号转导。vt.给出 为了提高关节中的溶质清除率，我们必须使用生物材料设计新的仓库式配方 提供像米安色林这样的小分子。聚乳酸-乙醇酸共聚物(PLGA)微球是一种用途广泛的 和生物相容的输送载体，非常适合将分子输送到关节，它们可以被设计成 通过靶向Rsp2信号通路给予米安色林持续的关节内cWnt通路抑制。 摘要目的：验证Rsp2在PTOA发病机制中的作用及持续性关节内治疗的疗效。 米安色林PLGA微球抑制Rsp2的研究。 具体目标：1)。2)阐明Rspo2在PTOA进展中的作用；推进……的发展 PLGA微球实现了小分子Rsp2抑制剂的体内缓释；3)测试其效果-- 持续关节内Rsp2抑制剂治疗作为一种新的PTOA治疗方法。 研究计划：目标1)。我们将描述Rspo2反应细胞的类型，并描述疾病的亲缘关系。 Rsp2对TCF/Lef Wnt报告小鼠健康关节和损伤关节的运动作用单细胞RNAseq将洗脱- Cate Rspo2-LGR信号转导细胞，同时无偏见地评估对Rsp2的转录反应。Rspo2将 然后在关节损伤时使用ROSA26-CreERT2；Rspo2f/f小鼠进行全局消融，PTOA严重程度将 被评估。目标2)。我们将研制米安色林PLGA微球，目标是在4周内 体外释放，爆发量可忽略不计。我们将确认4周的体内cWnt信号抑制并评估 安全。利用原代细胞，我们将研究Mianserin持续传递对cWnt信号转导的影响。 软骨细胞、滑膜成纤维细胞和骨祖细胞。我们将进一步测试基于PLGA的米安色林递送- 在全膝关节置换术中获得的经Rsp2处理的人软骨移植的ERY。目标3)。我们将管理- 米安色林负载PLGA微球到损伤后关节和综合表型PTOA， 对反复注射米安色林和反复注射β-连环蛋白抑制剂的疗效进行基准比较。我们会核实 米安色林的体内效应是通过在WT和WT中进行这些研究而抑制Rsp2而介导的 Rspo2条件性基因敲除小鼠，我们将使用IHC确认cWnt信号的抑制。

ABSTRACT INVESTIGATORS: Tristan Maerz, PhD (PI) is an ESI biomedical engineer focused on developing new treat- ments for post-traumatic osteoarthritis (PTOA). The Co-Investigators Kurt Hankenson, DVM PhD and Craig Duvall, PhD have expertise in Wnt signaling and biomaterial-mediated tissue regeneration, respectively. RESEARCH CONTEXT: Overactivation of canonical Wnt/β-Catenin (cWnt) signaling is increasingly recognized as a driver of joint degeneration in PTOA. A novel cWnt agonist in the context of PTOA is R-spondin 2 (Rspo2), which some literature and our data support as a disease-promoting ligand via cWnt-mediated induc- tion of synovial fibrosis, protease overexpression, chondrocyte hypertrophy, and aberrant bone sclerosis. A recent drug screen identified that the drug Mianserin inhibits Rspo2 signaling by blocking Lgr5 binding. Given high solute clearance rates in the joint, we must engineer new depot-type formulations using biomaterials to deliver small molecules such as Mianserin. Poly(lactic-co-glycolic acid) (PLGA) microspheres are a versatile and biocompatible delivery vehicle well-suited to deliver molecules to the joint, and they can be engineered to deliver Mianserin for sustained intra-articular cWnt pathway inhibition by targeting Rspo2 signaling. OBJECTIVE: To validate the role of Rspo2 in PTOA pathogenesis and test efficacy of sustained intra-articular Rspo2 inhibition using Mianserin-loaded PLGA microspheres. SPECIFIC AIMS: 1). Elucidate the impact of Rspo2 on PTOA progression; 2). Advance the development of PLGA microspheres to achieve sustained in vivo delivery of a small-molecule Rspo2 inhibitor; 3). Test the effi- cacy of sustained intra-articular Rspo2 inhibitor therapy as a novel PTOA treatment. RESEARCH PLAN: Aim 1). We will delineate Rspo2-responsive cell types and characterize the disease-pro- moting effect of Rspo2 in healthy and injured joints of TCF/LEF Wnt reporter mice. Single-cell RNAseq will elu- cidate Rspo2-Lgr signaling cells while unbiasedly assessing transcriptional responses to Rspo2. Rspo2 will then be ablated globally at the time of joint injury using Rosa26-CreERT2; Rspo2f/f mice and PTOA severity will be assessed. Aim 2). We will formulate Mianserin-loaded PLGA microspheres, aiming to achieve >4 weeks of in vitro release with negligible burst. We will confirm 4 weeks of in vivo cWnt signaling suppression and assess safety. Using primary cells, we will examine the effect of sustained Mianserin delivery on cWnt signaling in chondrocytes, synovial fibroblasts, and osteoprogenitor cells. We will further test PLGA-based Mianserin deliv- ery on Rspo2-treated human cartilage explants obtained during total knee arthroplasty. Aim 3). We will admin- ister Mianserin-loaded PLGA microspheres to the joint following injury and comprehensively phenotype PTOA, benchmarking efficacy against repeated Mianserin and repeated β-Catenin inhibitor injections. We will verify that the in vivo effect of Mianserin is mediated through Rspo2 inhibition by performing these studies in WT and Rspo2 conditional knockout mice, and we will confirm suppression of cWnt signaling using IHC.