Targeting PHLPP to treat interval disc degeneration using surgical and drug delivery methods

项目总结 痛性椎间盘退行性变(IDD)的迅速增加使其成为迫切需要发展的疾病 延缓IDD进展的解决方案。IDD与慢性炎症、IVD细胞丧失、 通过AKT、PKC和MAPK的去磷酸化加速基质降解和细胞凋亡 信号通路。我们提出的初步证据表明，磷酸酶Pleckstrin同源结构域 富含亮氨酸重复蛋白磷酸酶1(PHLPP1)促进IDD。因此，PHLPP1的全局删除 通过防止基质降解和减少促炎作用抑制老年小鼠IDD的进展 细胞因子的表达。我们对退化的人类NP细胞的初步数据表明，促炎因子 用小分子PHLPP抑制剂治疗后，反应降低。我们之前开发了 可注射的纳米配方，能够在延长的时间内以恒定的速度输送小分子 一段时间。基于这些发现，我们对概念和可行性的初步证明以及我们最近的 ，我们建议检验这一新的假设，即NP对PHLPP1的隔室特异性抑制将 抑制增龄性自发性炎症和基质降解延缓疾病进展 IDD。 目的1利用AGE诱导的模型，在体内确定PHLPP1在IDD中的作用。 条件性PHLPP1基因敲除小鼠的自发性IDD。PHLPP1将在NP(NPcKO)中枯竭，其作用 将通过免疫组织化学、分子和生物力学方法对PHLPP1进行评估。体外研究 将通过单细胞RNA测序和分子生物学来评估差异调控途径 小鼠NPcKO以及人NP细胞中PHLPP1基因敲除的分析。目标2将测试一种 小分子PHLPP抑制剂通过开发可注射的长期纳米制剂来减缓IDD 在自发性缺碘小鼠模型中释放PHLPP抑制剂并评价其疗效。 这个项目意义重大，因为在美国，椎间盘源性背痛是一种主要负担。新的 对IDD的机制和微创治疗的见解将结合机械性和平移性 学习。这项研究具有创新性，因为小分子PHLPP抑制剂的开发尚未得到 以及使用近红外标记的纳米制剂来控制PHLPP抑制剂的输送 治疗IDD是一项技术创新。这项研究的成功完成将为我们对NP提供新的见解 碘缺乏病的房室特异性进展及新型小分子PHLPP抑制剂载药纳米制剂 注射作为人类IDD的一种潜在的疾病修改治疗。

PROJECT SUMMARY The rapid increase in painful intervertebral disc (IVD) degeneration (IDD) makes it an urgent need to develop solutions for delaying IDD progression. IDD is associated with chronic inflammation, loss of IVD cellularity, matrix degradation and apoptosis, which are accelerated by dephosphorylation of AKT, PKC, and MAPK signaling pathways. We present preliminary evidence that the phosphatase Pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) promotes IDD. Thus global deletion of PHLPP1 repressed IDD progression in old mice by preventing matrix degradation and reducing pro-inflammatory cytokine expression. Our preliminary data on degenerated human NP cells suggested that pro-inflammatory responses were decreased after treatment with a small molecule PHLPP inhibitor. We previously developed injectable, nanoformulations that are capable of delivering small molecules at constant rates over an extended period of time. Based on these findings, our preliminary proof of concept and feasibility as well as our recent publication, we propose to test the novel hypothesis that NP compartment specific inhibition of PHLPP1 will delay disease progression via suppressing inflammation and matrix degradation in age-induced spontaneous IDD. Aim 1 will identify the role of PHLPP1 on IDD in the NP compartment in vivo using a model of age-induced spontaneous IDD in conditional Phlpp1 knockout mice. Phlpp1 will be depleted in NP (NPcKO) and the role of Phlpp1 will be assessed with immunohistochemical, molecular, and biomechanical methods. In vitro studies will evaluate the differentially regulated pathways by single cell RNA-sequencing and molecular-biological analysis of mouse NPcKO as well as PHLPP1 knockdown in human NP cells. Aim 2 will test the efficacy of a small molecule PHLPP inhibitor to decelerate IDD by developing an injectable nanoformulation for long-term PHLPP inhibitor release and evaluate its efficacy in a mouse model of spontaneous IDD. This project is highly significant because discogenic backpain is a major burden in the United States. New insights in mechanisms and minimal invasive treatments of IDD will combine mechanistic and translational studies. This study is innovative because the development of a small molecule PHLPP inhibitor has not been previously described and the use of NIR labeled nanoformulations for controlled PHLPP inhibitor delivery to treat IDD is a technical innovation. Successful completion of this study will provide new insights into NP compartment specific progression of IDD and advance small molecule PHLPP inhibitor laden nanoformulation injection as a potential disease-modifying treatment for IDD in humans.