Non-viral Reprogramming of Intervertebral Disc Cells for the treatment of Discogenic back pain

项目总结/摘要 慢性下背痛对社会造成重大的社会经济负担，并且是导致慢性下背痛的主要原因。 阿片类药物危机加剧去年，美国国立卫生研究院“推出了HEAL（帮助结束长期成瘾）计划， 积极的跨机构努力，加快科学解决方案，以遏制国家阿片类药物公共卫生危机”。 这种巨大的负担主要是因为研究未能针对相关的潜在机制 疼痛的产生。椎间盘（IVD）退行性变与腰椎间盘突出症的病理生理学密切相关。 下背痛和确定非成瘾性微创治疗椎间盘源性背痛（DBP）是一个 研究优先。病理性IVD变化包括细胞外基质（ECM）分解、炎症和炎症。 已经显示与疼痛显著相关的异常神经/血管向内生长。因此 DBP的最佳治疗将以结构恢复和减轻疼痛症状为目标。但目前 涉及使用干细胞或基因治疗的策略面临许多挑战，包括 干细胞无法适应苛刻的IVD微环境，使用病毒载体和不必要的DNA 宿主基因组内的缺失。此外，临床医生无法获得临床相关工具， 这些技术可以直接帮助治疗DBP患者的潜在疾病。迫切需要 寻找一种生物学上的非成瘾性策略来解决这些限制。我们的目标是利用新型细胞 重编程技术，以将天然患病IVD细胞的先天细胞表型改变为健康IVD细胞。 人体外细胞培养和体内细胞外产生和抗分解代谢/炎症表型 DBP模型用于将遗传货物转运到细胞中的非病毒递送，例如工程化的细胞外 囊泡（EV）或组织纳米转染（TNT）提供了安全和微创的重编程方法 体细胞和最近的工作研究人员已经证明成功的重编程成人 成纤维细胞转化为内皮细胞的体内小鼠模型。我们建议使用这些创新的非病毒递送 在体外和体内将遗传货物递送至IVD的系统。第一个具体目标（R61第1阶段目标1）侧重于 EV或TNT递送转录因子对患病的人髓核细胞的作用， 体外组织检查ECM和分解代谢、炎症和疼痛标记物的变化。第二个目标（R61 阶段1目的2）研究EV或TNT递送转录因子在小鼠IVD穿刺中的作用 评估椎间盘结构/功能、疼痛、认知和细胞毒性变化的DBP模型。这些研究 都是重要的和高度创新的，因为他们结合了联合收割机独特的多学科医疗团队， 和兽医临床医生，脊柱生物学家，神经科学家，生物医学工程师和生物统计学家， 询问使用这些新的概念和技术来治疗DBP。更广泛的影响 建议和过渡到R33部分涉及评估该策略的临床相关性 软骨营养不良的狗自发地发展DBP，随后在患有DBP的狗中进行临床试验。

Project Summary/Abstract Chronic low back pain exerts a significant socio-economic burden on society and is a major contributor to the growing opioid crisis. Last year, NIH “launched the HEAL (Helping to End Addiction Long-term) initiative, an aggressive, trans-agency effort to speed scientific solutions to stem the national opioid public health crisis”. This enormous burden is largely because studies have failed to target the underlying mechanisms associated with pain generation. Intervertebral disc (IVD) degeneration is strongly associated with the pathophysiology of low back pain and identifying non-addictive minimally invasive treatments for discogenic back pain (DBP) is a research priority. Pathological IVD changes include extracellular matrix (ECM) breakdown, inflammation and aberrant nerve/vascular ingrowth which have been shown to significantly correlate with pain. Therefore the optimal therapy for DBP would target both structural restoration and reduce the symptoms of pain. Yet current strategies involving the use of stem cells or gene therapy are faced with a number of challenges which include failure of stem cells to adapt to the harsh IVD microenvironment, the use of viral vectors and unwarranted DNA deletions within the host genome. Furthermore clinicians do not have access to clinically relevant tools or technologies that could directly help treat the underlying disease in patients with DBP. There is a critical need for a biological non-addictive strategy that addresses these limitations. Our goal is to use novel cellular reprogramming technologies to alter the innate cell phenotype of native diseased IVD cells to a healthy extracellular producing and anti-catabolic/inflammatory phenotype in human in vitro cell culture and in vivo models of DBP. Non-viral delivery for transporting genetic cargo into the cell such as engineered extracellular vesicles (EVs) or tissue nano-transfection (TNT) offer safe and minimally invasive methods for reprogramming somatic cells and recent work by the investigators has demonstrated successful reprogramming of adult fibroblasts into endothelial cells in vivo mouse models. We propose using these innovative non-viral delivery systems to deliver genetic cargo to IVDs in vitro and in vivo. The first specific aim (R61 Phase 1 Aim 1) focuses on the effects of EV or TNT delivery of transcription factors on diseased human nucleus pulposus cells and tissue in vitro examining changes in ECM and catabolic, inflammatory and pain markers. The second aim (R61 Phase 1 Aim 2) investigates the effects of EV or TNT delivery of transcription factors in mouse IVD puncture models of DBP assessing changes in disc structure/function, pain, cognition and cytotoxicity. These studies are both significant and highly innovative because they combine a unique multi-disciplinary team of medical and veterinary clinicians, spine biologists, neuroscientists, biomedical engineers, and a biostatistician to interrogate the use of these novel concepts and technologies to treat DBP. The broader impacts of this proposal and transition to the R33 portion involve assessing this strategy in clinically relevant chondrodystrophic dogs that develop DBP spontaneously followed by clinical trials in patient dogs with DBP.