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

基本信息

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
项目类别：
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

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

展开

项目摘要

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.

项目摘要退化性椎间盘疾病（DDD）是腰痛的主要来源，这不仅是主要原因活动限制和工作缺席，但也代表了社会的巨大财务燃烧。 DDD是定义为有症状的椎间盘（IVD）变性，明显分解代谢和炎症回答。炎症介质在DDD期间在疼痛发展中起着必不可少的作用。作为最新 DDD的治疗策略是有限的，通常与高风险有关，并且并不总是持久的，很棒由于其再生和抗炎潜力，希望曾经将希望固定在干细胞上。然而，各种研究（包括我们的）表明，基于间充质干细胞（MSC）的成功 DDD的处理受到限制，这是由于变性的IVD的严格微环境受阻，使细胞受阻。生存和功能。最近，已经提出了从MSC衍生的细胞外蔬菜（EV）作为DDD的潜在替代处理。重要的是，作为一种细胞的方法，生存挑战消除了与基于细胞的疗法相关的。电动汽车是从细胞中释放出的脂质膜颗粒作为纳米载体的功能，当源自MSC时，被认为具有再生能力。目标 MSC的遗传修饰，具有TNFα刺激的基因-6（TSG6）的过表达，可能会促进释放的电动汽车的再生能力通过改变其内容。 TSG6特别有前途表达已被确定为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颖，非阿片类药物，疼痛管理疗法铺平道路患有DDD的患者。具体而言，该项目将确定不同EV亚群的潜力从MSC释放，该MSC经历了TSG6的CRISPR/CAS9激活。我们将通过：（1）来解决这个目标建立TSG6修饰的MSC线并表征细胞并释放EV；（2）评估效果来自TSG6修饰的MSC的不同电动汽车大小亚群（这可能有所不同货物/吸收/迁移率）在退化的圆盘细胞上。我们将通过评估促炎细胞因子，分解代谢酶，神经因子和基质蛋白的表达。我们将结合两个最近的生物技术发展，并具有出色的治疗潜力（MSC衍生电动汽车和CRISPR/CAS9基因组工程）与隔离电动汽车亚群的技术事故（Nanopocket膜）。使用CRISPR/CAS9改善MSC衍生电动汽车的货物的概念改进的DDD处理是高度新颖的。如果成功，它有望提供一种与DDD作斗争的新疗法。这拟议项目的影响很大，因为迫切需要DDD的新，有针对性和非阿片类药物治疗方案因此，由于其高风险/高增益性质，患者非常适合R21机制。而且，结果发展可能会在其他退行性炎症疾病中找到应用。