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.

项目概要 退行性椎间盘疾病（DDD）是腰痛的主要原因，这不仅是腰痛的主要原因 活动限制和工作缺勤也给社会带来了巨大的经济负担。 定义为有症状的椎间盘 (IVD) 退变，伴有明显的分解代谢和炎症 目前，炎症介质在 DDD 的疼痛发展中发挥着不可或缺的作用。 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、有效的。 由于干细胞的再生和抗炎潜力，人们一度将希望寄托在干细胞上。 各种研究（包括我们的研究）已经证明基于间充质干细胞（MSC）的成功 由于退化的 IVD 的恶劣微环境阻碍了细胞的生长，DDD 的治疗受到限制 最近，有人提出源自 MSC 的细胞外囊泡 (EV)。 作为 DDD 的潜在替代疗法，重要的是，作为一种非细胞方法，生存面临着挑战。 与基于细胞的疗法相关的EV是从细胞中释放的脂质膜颗粒。 作为纳米载体，当源自间充质干细胞时，被认为具有靶向再生能力。 对 MSC 进行基因修饰，过度表达 TNFα 刺激基因 6 (TSG6)，可能会促进 通过改变 TSG6 的含量，所发布的电动汽车的再生能力特别有前途。 表达已被确定为 MSC 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型非阿片类药物疼痛管理疗法铺平道路 具体来说，该项目将确定不同 EV 亚群的潜力。 从经过 CRISPR/Cas9 激活 TSG6 的 MSC 中释放出来，我们将通过以下方式实现这一目标：(1) 建立TSG6修饰的MSCs系并表征细胞和释放的EV（2）评估效果； 来自 TSG6 修饰的 MSC 的不同 EV 大小的亚群（可能在 我们将通过以下方式确定这些 EV 的治疗潜力。 评估促炎酶细胞因子、分解代谢酶、神经因子和基质蛋白的表达。 我们将结合两项具有突出治疗潜力的最新生物技术发展（MSC 衍生 EV 和 CRISPR/Cas9 基因组工程）以及用于分离 EV 亚群的技术发明 （纳米袋膜）使用 CRISPR/Cas9 改善 MSC 衍生的 EV 的载量的概念。 改进的 DDD 治疗方法非常新颖，如果成功，它有望提供一种对抗 DDD 的新疗法。 由于 DDD 迫切需要新的、有针对性的非阿片类药物治疗方案，因此拟议的项目具有影响力 患者，因此，由于其高风险/高收益的性质，非常适合 R21 机制。 其进展可能会在其他退行性炎症疾病中得到应用。