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

摘要 Tristan Maerz博士（PI）是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）。共同研究者Kurt Hankenson，DVM博士和克雷格 杜瓦尔博士分别在Wnt信号传导和生物材料介导的组织再生方面具有专长。 研究背景：经典Wnt/β-连环蛋白（cWnt）信号转导的过度激活越来越被认识到 作为PTOA关节退化的驱动因素。PTOA背景下的新型cWnt激动剂是R-spondin 2 （Rspo 2），一些文献和我们的数据支持其作为一种疾病促进配体，通过cWnt介导的诱导， 滑膜纤维化、蛋白酶过度表达、软骨细胞肥大和异常骨硬化的作用。一 最近的药物筛选鉴定出药物米安色林通过阻断Lgr 5结合来抑制Rsp 〇 2信号传导。给定 由于关节中的高溶质清除率，我们必须使用生物材料设计新的贮库型制剂， 释放小分子物质如米安色林聚（乳酸-羟基乙酸）（PLGA）微球是一种多用途的 和生物相容性递送载体，非常适合于将分子递送到关节，并且它们可以被设计成 通过靶向Rspo 2信号传导递送米安色林用于持续的关节内cWnt通路抑制。 目的：验证Rspo 2在PTOA发病机制中的作用，并测试持续关节内注射的疗效。 使用米安色林负载的PLGA微球的Rspo 2抑制。 具体目标：1）。阐明Rspo 2对PTOA进展的影响; 2）.促进这些国家发展 PLGA微球以实现小分子Rspo 2抑制剂的持续体内递送; 3）.测试效果- 持续关节内Rspo 2抑制剂治疗作为一种新型PTOA治疗的可行性。 研究目标：目标1）。我们将描述Rspo 2反应细胞类型，并描述疾病前 Rspo 2在TCF/LEF Wnt报告小鼠的健康和损伤关节中的运动作用。单细胞RNAseq将elu- cidate Rspo 2-Lgr信号传导细胞，同时无偏地评估对Rspo 2的转录应答。Rspo 2将 然后在关节损伤时使用Rosa 26-CreERT 2; Rspo 2f/f小鼠整体消融，并且PTOA严重程度将 进行评估。目标2）。我们将配制装载米安色林的PLGA微球，旨在实现>4周的 体外释放，爆发可忽略不计。我们将确认4周的体内cWnt信号传导抑制，并评估 安全为代价的使用原代细胞，我们将检查持续的米安色林递送对细胞中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的米安色林递送， 在全膝关节置换术中获得的Rspo 2处理的人软骨外植体上进行研究。目标3）。我们将管理- 米安色林PLGA微球对关节损伤后综合表型PTOA的影响， 对重复米安色林和重复β-连环蛋白抑制剂注射的基准功效进行比较。我们将核实 通过在WT中进行这些研究，米安色林的体内作用是通过Rspo 2抑制介导的， Rspo 2条件性敲除小鼠，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.