Novel non-viral reprogramming strategies to treat Discogenic back pain via engineered extracellular vesicles

项目总结/摘要 慢性腰痛是一种具有重要社会经济意义的衰弱性疾病， 阿片类药物的使用因此，旨在通过靶向治疗疼痛的微创非成瘾性治疗方法， 潜在的疾病病理学对于改善人类健康和限制日益严重的阿片类药物危机至关重要。 椎间盘退变与慢性腰痛的病理生理学密切相关; 特别是细胞外基质（ECM）分解、炎症和异常神经/血管向内生长， 这与“椎间盘源性背痛”（DBP）显著相关。因此，这一总体目标 一项提议是开发新的基于非病毒重编程的疗法， 髓（NP）和纤维环（AF）IVD细胞与DBP相关，转化为健康的促合成代谢， 抗神经/血管表型，使用工程化的细胞外囊泡（eEV）。我们的核心假设是 非病毒重编程将恢复IVD结构/机械功能，并限制神经/血管向内生长 通过将患者自身退化的IVD细胞原位转化为促合成代谢表型， 迄今为止，临床医生无法获得必要的生物工具来治疗患者的IVD变性 关于DBP当前生物学策略成功的关键障碍涉及大量的后勤和 监管挑战，如缺乏持续的药物递送系统，长期细胞活力差或病毒感染， 重新编程，永久地与宿主DNA整合。我们的非成瘾策略侧重于 解决这些局限性。通过我们最近的R61奖和发表的工作，我们已经表明，人类 NP细胞可以使用发育转录因子重编程为更健康的表型 Brachyury或FOXF 1，显示ECM积累增加和分解代谢/炎性/炎症/炎症减少。 神经营养因子-健康IVD的所有关键特征。此外，在我们的小鼠DBP体内模型中， 也证明了在动物中NP组织水合作用和疼痛行为的抑制方面的显著改善 用装载FOXF 1的eEV治疗长达12周，突出了我们提出的策略的潜力， 恢复IVD的结构/功能，同时减轻疼痛。然而，关键的差距仍然存在，如i）理解 NP和AF细胞中多种发育转录因子的协同重编程潜力，ii） 性别和年龄如何影响我们的策略，以及iii）使用更临床相关的 模拟人类状况和功能化eEV以递送转录因子的体内动物模型 IVD内的特定细胞类型（例如，NP或AF细胞）。 我们的第一个目标是研究负载有多种转录因子的eEV的协同作用， DBP的体外和体内临床相关模型，并通过评估IVD量化我们策略的疗效 结构/功能和疼痛标志物。第二个目的涉及用对以下特异性的配体官能化eEV： NP或AF细胞以在体外和体内选择性地将货物递送至IVD的简并NP或AF细胞。

Project Summary/Abstract Chronic low back pain is a debilitating disorder of significant socio-economic importance and a major gateway to opioid use. Therefore, minimally invasive non-addictive treatments that aim to address pain by targeting the underlying disease pathology are critical to improve human health and limit the growing opioid crisis. Intervertebral disc (IVD) degeneration is strongly associated with the pathophysiology of chronic low back pain; specifically extracellular matrix (ECM) breakdown, inflammation, and aberrant nerve/vascular ingrowth, all of which are significantly correlated with “Discogenic back pain” (DBP). Therefore, the overall objective of this proposal is to develop novel non-viral reprogramming-based therapies to convert degenerate nucleus pulposus (NP) and annulus fibrosus (AF) IVD cells associated with DBP, into a healthy pro-anabolic, anti-nerve/vascular phenotype, using engineered extracellular vesicles (eEVs). Our central hypothesis is that non-viral reprogramming will restore IVD structure/mechanical function, and limit nerve/vascular ingrowth associated with pain, by converting the patient’s own degenerate IVD cells into a pro-anabolic phenotype in situ. To date, clinicians do not have access to the necessary biological tools to treat IVD degeneration in patients with DBP. A critical barrier to the success of current biologic strategies involves significant logistical and regulatory challenges such as a lack of sustained drug delivery systems, poor long-term cell viability or viral reprogramming that permanently integrates with the host DNA. Our non-addictive strategy focuses on addressing these limitations. Through our recent R61 award and published work, we have shown that human NP cells can be reprogrammed towards a healthier phenotype using developmental transcription factors Brachyury or FOXF1, showing increased ECM accumulation and decreased catabolic/ inflammatory/ neurotrophic factors - all key features of a healthy IVD. Furthermore, in our mouse DBP in vivo model we have also demonstrated significant improvement in NP tissue hydration and dampened pain behaviors in animals treated with eEVs loaded with FOXF1 for up-to 12 weeks, highlighting the potential of our proposed strategy to restore structure/function of the IVD while reducing pain. However, critical gaps remain such as i) understanding the synergistic reprogramming potential of multiple developmental transcription factors in NP and AF cells, ii) how sex and age influence our strategy, and iii) evaluating therapeutic efficiency using more clinically relevant in vivo animal models that simulate the human condition and functionalized eEVs to deliver transcription factors to specific cell types within the IVD (for example, NP or AF cells). Our first aim investigates the synergistic effects of eEVs loaded with multiple transcription factors using in vitro and in vivo clinically relevant models of DBP, and quantifying efficacy of our strategy via assessment of IVD structure/function and pain markers. The second aim involves functionalizing the eEVs with ligands specific for NP or AF cells to selectively deliver cargo to degenerate NP or AF cells of the IVD in vitro and in vivo.