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

摘要 调查员：Tristan Maerz 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。联合研究人员 Kurt Hankenson（DVM 博士）和 Craig Duvall 博士分别在 Wnt 信号传导和生物材料介导的组织再生方面拥有专业知识。 研究背景：人们越来越认识到经典 Wnt/β-Catenin (cWnt) 信号传导的过度激活 作为 PTOA 关节退化的驱动因素。 PTOA 中的一种新型 cWnt 激动剂是 R-spondin 2 (Rspo2)，一些文献和我们的数据支持其通过 cWnt 介导的诱导作用作为促进疾病的配体 滑膜纤维化、蛋白酶过度表达、软骨细胞肥大和异常骨硬化。一个 最近的药物筛选发现，药物 Mianserin 通过阻断 Lgr5 结合来抑制 Rspo2 信号传导。给定 由于关节中的溶质清除率较高，我们必须使用生物材料设计新的储库型配方，以 输送小分子，例如米安色林。聚乳酸-乙醇酸 (PLGA) 微球是一种多功能的 和生物相容性输送载体非常适合将分子输送到关节，并且它们可以被设计为 通过靶向 Rspo2 信号传导，递送米安色林以持续抑制关节内 cWnt 通路。 目的：验证Rspo2在PTOA发病机制中的作用并测试持续关节内注射的疗效。 使用负载 Mianserin 的 PLGA 微球抑制 Rspo2。 具体目标： 1).阐明Rspo2对PTOA进展的影响； 2）。推进发展 PLGA微球实现小分子Rspo2抑制剂的持续体内递送； 3）。测试效率 持续关节内 Rspo2 抑制剂治疗作为新型 PTOA 治疗的有效性。 研究计划：目标 1)。我们将描述 Rspo2 反应细胞类型并表征疾病亲 Rspo2 对 TCF/LEF Wnt 报告小鼠健康和受伤关节的运动作用。单细胞 RNAseq 将洗脱 cidate Rspo2-Lgr 信号细胞，同时公正地评估对 Rspo2 的转录反应。 Rspo2 将 然后在关节损伤时使用 Rosa26-CreERT2 进行全局消融； Rspo2f/f 小鼠和 PTOA 严重程度将 进行评估。目标2）。我们将配制负载 Mianserin 的 PLGA 微球，旨在实现 >4 周的效果 体外释放可忽略不计。我们将确认 4 周的体内 cWnt 信号传导抑制并评估 安全。使用原代细胞，我们将检查持续递送米安色林对 cWnt 信号传导的影响 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 Mianserin 交付 在全膝关节置换术中获得的经 Rspo2 处理的人类软骨外植体的研究。目标3）。我们将管理- 将载有 Mianserin 的 PLGA 微球注射到损伤后的关节并进行综合表型 PTOA， 与重复注射米安色林和重复注射 β-连环蛋白抑制剂进行基准疗效比较。我们将核实 通过在 WT 和 Rspo2 条件敲除小鼠，我们将使用 IHC 确认 cWnt 信号传导的抑制。

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.