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

CRISPR 激活的 MSC 产生的细胞外囊泡：退行性椎间盘疾病的潜在疗法

• 批准号: 10733029
• 负责人: Karin Wuertz-Kozak
• 金额: $ 20.15万
• 依托单位: ROCHESTER INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10733029
• 关键词: Adipose tissue; Affect; Anti-Inflammatory Agents; Antiinflammatory Effect; Back Pain; Biological; Biological Assay; Biotechnology; CRISPR-mediated transcriptional activation; CRISPR/Cas technology; Catabolism; Cell Line; Cell Survival; Cell Therapy; Cell physiology; Cell surface; Cells; Chemistry; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Degenerative polyarthritis; Development; Direct Costs; Disease; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Exploratory/Developmental Grant; Exposure to; Financial Hardship; Future; Gene Expression Profiling; Genes; Genetic; Genome engineering; Glucose; Goals; Hand; Hip region structure; Human; Impairment; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Intervertebral disc structure; Knee; Low Back Pain; Mediator; Membrane; Membrane Lipids; Mesenchymal Stem Cells; Methods; Minor; Modification; Natural regeneration; Nature; Neck; Neck Pain; Nerve; Osmolar Concentration; Outcome; Pain; Pain management; Pathology; Patients; Persons; Play; Process; Production; Property; Proteins; Proteomics; Public Health; Publishing; Regenerative capacity; Reproducibility; Research; Research Personnel; Role; Societies; Source; Surface; TNF gene; Technology; Testing; Therapeutic; Therapeutic Effect; Tissues; Transmission Electron Microscopy; Ultracentrifugation; United States; Western Blotting; Work; alternative treatment; autocrine; cell behavior; clinical translation; combat; cytokine; exosome; extracellular vesicles; high risk; improved; innovation; intervertebral disk degeneration; invention; microvesicles; minimally invasive; nanocarrier; nanoparticle; non-opioid analgesic; novel; novel therapeutics; nucleus pulposus; overexpression; particle; patient population; protein expression; regenerative; stem cell therapy; stem cells; success; transcription factor; transcriptomics; treatment strategy; uptake; vesicular release

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.

项目总结 退行性腰椎间盘疾病(DDD)是腰痛的一个主要来源，它不仅是导致腰痛的主要原因 活动受限和工作缺勤，也给社会带来了巨大的经济负担。DDD为 定义为有明显分解代谢和炎症的症状性腰椎间盘(IVD)变性 回应。炎症介质在DDD期间的疼痛发展中起着不可或缺的作用。当前状态 DDD的治疗策略是有限的，往往与高风险相关，而且并不总是持久的、伟大的 干细胞由于具有再生和抗炎的潜力，一度被寄予希望。然而， 各种研究(包括我们的研究)已经证明，以间充质干细胞(MSC)为基础的成功 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 生存和功能。最近，有人提出了来源于间充质干细胞的细胞外小泡(Ev)。 作为治疗DDD的一种潜在替代疗法。重要的是，作为一种非细胞方法，生存挑战 与以细胞为基础的治疗相关的疾病被消除。EVS是从细胞释放的脂膜颗粒， 作为纳米载体发挥作用，当从MSCs衍生时，被认为具有再生能力。目标明确 过表达肿瘤坏死因子α刺激基因-6(TSG6)的骨髓间充质干细胞的基因修饰可能促进 通过改变发布的电动汽车的内容来实现再生能力。TSG6特别有希望成为它的 表达被认为是骨髓间充质干细胞再生和组织保护能力的关键调节因子。 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。具体地说，该项目将确定不同电动汽车亚群的潜力 从经历了TSG6的CRISPR/Cas9激活的MSCs释放出来。我们将通过以下方式实现这一目标：(1) 建立TSG6修饰的间充质干细胞系，并对细胞和释放的EVS进行鉴定；(2)评价其作用 来自TSG6修饰的MSCs的不同EVS大小的亚群(可能在 货物/摄取/移动)在退化的盘细胞上。我们将通过以下方式确定这些电动汽车的治疗潜力 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。 我们将结合两个最新的生物技术发展与突出的治疗潜力(MSC衍生 EVS和CRISPR/Cas9基因组工程)和一项分离EV亚群的技术发明 (纳米囊膜)。利用CRISPR/CAS9改善MSC衍生电动汽车货运量的概念 改进的DDD治疗是非常新颖的。如果成功，它有望提供一种对抗DDD的新疗法。这个 由于迫切需要治疗DDD的新的、有针对性的和非阿片类药物治疗方案，拟议的项目具有影响力 因此，R21机制非常适合R21机制，因为它具有高风险/高收益的性质。此外，结果 这一进展可能会应用于其他退行性炎症性疾病。