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

生物材料介导的 R-spondin 2 持续抑制对创伤后骨关节炎中病理性 Wnt 信号传导的影响

基本信息

批准号：
10704167
负责人：
Tristan Maerz
金额：
$ 52.07万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2027-06-30
项目状态：
未结题

项目摘要

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.

抽象的研究人员：Tristan Maerz博士（PI）是一名ESI生物医学工程师，致力于开发新的治疗创伤后骨关节炎（PTOA）的生物。共同研究者Kurt Hankenson，DVM博士和Craig Duvall，PhD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业知识。研究环境：越来越多地认识到规范Wnt/β-catenin（CWNT）信号的过度激活作为PTOA联合变性的驱动力。 PTOA背景下的一种新型CWNT激动剂是R-Spondin 2 （RSPO2），一些文献和我们的数据支持作为促进疾病的配体通过CWNT介导的诱导 - 滑膜纤维化，蛋白酶过表达，软骨细胞肥大和异常骨硬化的影响。一个最近的药物筛查表明，药物甲蛋白酶通过阻断LGR5结合抑制RSPO2信号传导。给出在关节中，我们必须使用生物材料来设计新的仓库型公式传递小分子，例如硅蛋白酶。聚（乳酸 - 乙醇酸）（PLGA）微球是一种用途和生物相容性的送货车辆非常适合将分子传递到关节，可以设计为通过靶向RSPO2信号传导，输送硅蛋白酶以持续的关节内CWNT途径抑制。目的：验证RSPO2在PTOA发病机理中的作用和持续关节内的测试效率使用甲素蛋白负载的PLGA微球抑制RSPO2。具体目的：1）。阐明RSPO2对PTOA进展的影响； 2）。推进发展 PLGA微球以实现小分子RSPO2抑制剂的持续体内递送； 3）。测试努力 - 持续的关节内RSPO2抑制剂疗法作为一种新型PTOA治疗。研究计划：目标1）。我们将描述RSPO2响应性细胞类型，并表征疾病 - 疾病pro- RSPO2在TCF/LEF WNT报告的健康和受伤关节中的运动效应。单细胞rnaseq将elu- 确定RSPO2-LGR信号传导细胞，同时无偏评估对RSPO2的转录响应。 RSPO2会然后使用Rosa26-Creert2在关节损伤时在全球扫描； RSPO2F/F小鼠和PTOA严重程度将被评估。目标2）。我们将制定负载甲蛋白的PLGA微球，旨在达到> 4周的体外释放，爆发微不足道。我们将确认4周的体内CWNT信号抑制和评估安全。使用原代细胞，我们将检查持续的米亚梅林递送对CWNT信号传导的影响软骨细胞，滑膜成纤维细胞和骨源细胞。我们将进一步测试基于PLGA的Mianserin deliv- 在全膝关节置换术期间获得的RSPO2处理的人软骨外植体。目标3）。我们将管理损伤后，iSter甲素蛋白负载的PLGA微球对关节和全面表型PTOA，基准测试效率针对重复的硅脂和反复的β-catenin抑制剂注射。我们将验证通过在WT和 RSPO2有条件的敲除小鼠，我们将使用IHC确认抑制CWNT信号传导。