Extracellular vesicles produced by CRISPR-activated MSCs: A potential therapy for degenerative disc disease

项目摘要 退行性椎间盘疾病（DDD）是下腰痛的主要来源，不仅是下腰痛的主要原因， 活动限制和工作缺勤，而且也给社会带来巨大的财政负担。ddd是 定义为症状性椎间盘（IVD）退变，伴有明显的分解代谢和炎症 应答炎症介质在DDD期间的疼痛发展中起着不可或缺的作用。为流动 DDD的治疗策略是有限的，通常与高风险相关，并且并不总是持久的， 由于干细胞具有再生和抗炎的潜力，人们一度将希望寄托在干细胞上。然而，在这方面， 各种研究（包括我们的）已经证明，基于间充质干细胞（MSC）的成功 DDD的治疗是有限的，因为退化的IVD的恶劣微环境阻碍了细胞增殖， 生存和功能。最近，已经提出了来源于MSC的细胞外囊泡（EV 作为DDD的潜在替代治疗。重要的是，作为一种非细胞方法， 与基于细胞的疗法相关的疾病被消除。EV是从细胞释放的脂质膜颗粒， 作为纳米载体发挥作用，并且当来源于MSC时，被认为具有再生能力。针对性 通过过度表达TNF α刺激基因6（TSG6）对MSCs进行遗传修饰， 通过改变它们的内容，释放电动汽车的再生能力。TSG6特别有前途，因为它 表达已被鉴定为MSC的再生和组织保护能力的关键调节剂。 这项研究的长期目标是为新的，非阿片类药物的疼痛管理疗法铺平道路。 患有DDD的患者。具体来说，该项目将确定不同EV亚群的潜力 从经历TSG6的CRISPR/Cas9活化的MSC释放。我们将通过以下方式实现这一目标：（1） 建立TSG6基因修饰的MSCs细胞系，并对细胞和释放的EV进行表征;（2）评价TSG6基因修饰的MSCs的效果。 来自TSG6修饰的MSC的不同EV大小的亚群（其可能在 货物/摄取/移动性）。我们将通过以下方式确定这些EV的治疗潜力： 评估促炎细胞因子、分解代谢酶、神经因子和基质蛋白的表达。 我们将结合联合收割机两个最近的生物技术发展与突出的治疗潜力（MSC衍生的 EVs和CRISPR/Cas9基因组工程），并具有用于分离EV亚群的技术发明 （纳米袋膜）。使用CRISPR/Cas9来改善MSC衍生的EV的货物的概念， 改进的DDD治疗是非常新颖的。如果成功，它有望提供一种新的治疗方法来对抗DDD。的 由于迫切需要新的、有针对性的和非阿片类药物治疗DDD，因此拟议的项目具有影响力 患者，因此，由于其高风险/高收益性质，很好地适合R21机制。此外，结果 并且开发可能在其他变性炎症疾病中找到应用。

PROJECT SUMMARY Degenerative disc disease (DDD) is a major source of low back pain, which is not only the leading cause of activity limitation and work absence but also represents a tremendous financial burden to the society. DDD is defined as symptomatic intervertebral disc (IVD) degeneration with pronounced catabolic and inflammatory responses. Inflammatory mediators play an indispensable role in pain development during DDD. As current treatment strategies for DDD are limited, often associated with high risks, and are not always long-lasting, great hopes were once pinned on stem cells due to their regenerative and anti-inflammatory potential. However, various studies (including ours) have demonstrated that the success of mesenchymal stem cell (MSC)-based treatments for DDD is limited due to the harsh microenvironment of the degenerated IVD that hampers cell survival and functionality. More recently, extracellular vesicles (EVs) derived from MSCs have been suggested as a potential alternative treatment for DDD. Importantly, as an acellular approach, survival challenges associated with cell-based therapies are eliminated. EVs are lipid membrane particles released from cells that function as nanocarriers and, when derived from MSCs, are thought to have regenerative capacity. Targeted genetic modification of MSCs, with overexpression of TNFα-stimulated gene-6 (TSG6), may promote the regenerative capacity of the released EVs by altering their content. TSG6 is specifically promising as its expression has been identified as a crucial regulator of the regenerative and tissue-protective capacity of MSCs. The long-term goal of this research is to pave the way toward novel, non-opioid, pain management therapies for patients suffering from DDD. Specifically, this project will determine the potential of different EV subpopulations released from MSCs that underwent CRISPR/Cas9 activation of TSG6. We will address this goal by: (1) Establishing the TSG6-modified MSCs line and characterizing cells and released EVs; (2) Evaluating the effect of different EVs size subpopulations derived from TSG6-modified MSCs (which may differ in cargo/uptake/mobility) on degenerated disc cells. We will determine the therapeutic potential of these EVs by evaluating the expression of proinflammatory cytokines, catabolic enzymes, nerve factors, and matrix proteins. We will combine two recent biotechnological developments with outstanding therapeutic potential (MSC-derived EVs and CRISPR/Cas9 genome engineering) with a technical invention for the isolation of EV subpopulations (nanopocket membrane). The concept of using CRISPR/Cas9 to improve the cargo of MSC-derived EVs for improved DDD treatment is highly novel. If successful, it promises to provide a new therapy to combat DDD. The proposed project is impactful due to the urgent need for new, targeted, and non-opioid treatment options for DDD patients, and hence, fits the R21 mechanism well because of its high risk/high gain nature. Moreover, results and developments are likely to find applications in other degenerative-inflammatory diseases.