Systemic delivery of muscle stem cell for muscle disease therapy

肌肉干细胞的全身递送用于肌肉疾病治疗

• 批准号: 10615789
• 负责人: ATSUSHI ASAKURA
• 金额: $ 17.05万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10615789
• 关键词: Achievement; Affect; Animal Model; Autologous; B-Lymphocytes; Blood Circulation; Capillary Endothelial Cell; Cell Nucleus; Cell Separation; Cell Survival; Cell Therapy; Cell Transplantation; Cells; Chronic; Circulation; Clinical Trials; Coculture Techniques; Code; Communication; Disease; Duchenne muscular dystrophy; Dystrophin; Encapsulated; Engraftment; Extravasation; Failure; Genes; Genetic; Goals; Human; Immune; Injections; Intra-Arterial Injections; Intramuscular Injections; Lipids; Mannitol; Mediating; Mediator; Membrane; Methods; Microscope; Modification; Mus; Muscle; Muscle Contraction; Muscle Fibers; Muscle satellite cell; Muscular Dystrophies; Mutation; Myoblasts; Myopathy; Nucleic Acids; Optics; Outcome; Patients; Proliferating; Proteins; Protocols documentation; Quality of life; Research; Resolution; Sarcolemma; Scheme; Skeletal Muscle; Skeletal Muscle Satellite Cells; Source; Stem cell transplant; Structure; System; Testing; Therapeutic; Thinness; Three-Dimensional Imaging; Tissues; Transduction Gene; Transfection; Transgenes; Transplantation; Vascular Permeabilities; cell type; cremaster muscle; extracellular vesicles; improved; induced pluripotent stem cell; injured; insight; mdx mouse; micro-dystrophin; migration; muscle degeneration; muscle regeneration; muscular dystrophy mouse model; nanoparticle; novel therapeutic intervention; overexpression; practical application; precursor cell; prevent; programs; real-time images; receptor; repaired; restoration; satellite cell; scale up; self-renewal; stem cells; vector

Abstract Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene coding for dystrophin, which functions to maintain muscle fiber structure and function in the whole body by preventing it from being damaged during muscle contraction. Presently, there is no definitive treatment for DMD patients, and current therapies focus on prolonging survival and improving quality of life. Definitive treatment will require that functional dystrophin protein is restored in all affected muscle groups. Possible approaches to restoring dystrophin expression in muscle fibers include cell therapy.We hope that transplantation of a particular type of cell capable of regenerating muscle will help us to develop new therapeutic approaches to this disease. Muscle stem cells, termed satellite cells, isolated from healthy donors or patients should be able to provide dystrophin and repair muscle damage in DMD patients. For efficient DMD therapy, satellite cells which maintain the ability to self-renew are also necessary. However, satellite cells are rare (2-7% of all muscle nuclei) and often difficult to isolate. In addition, efficient myogenic differentiation and the scale-up of myogenic differentiation remain elusive and must be developed further in order to generate effective cell-based therapy for chronic muscle diseases. Patient-derived induced pluripotent stem cells (iPSCs) are the ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. However, the failure of systemic delivery of the injected cells has hindered practical application in patients. Our long-term goals are to develop an effective supply of satellite cells for cell therapy, to find an efficient systemic delivery method that can treat all affected muscles, and to enable the injected cells to self-renew and reach an effective mass. The specific aims of this application: 1. To determine possible interactions between CD24-expressing satellite cells and capillary endothelial cells via real-time imaging and 3- D imaging after our recently established whole muscle tissue clearing protocol. 2. To develop systemic delivery methods for satellite cells and hiPSC-derived myogenic progenitor cells in combination with genetic modification, including transduction of an extravasation factor, CD24, and Mannitol, an agent for enhancing vascular permeabilization. 3. To develop systemic delivery methods for satellite cells via Extracellular Vesicle (EV)- mediated CD24 transfer. In combination with gene and protein transduction, this concerted approach will help us to make satellite cells that can be systemically transplanted into patients for a definitive cure of DMD. The proposed specific aims will test whether systemically-injected satellite cells can be delivered to a target injured muscle and contribute to muscle regeneration as well as to the satellite cell pool. The anticipated outcome of the proposed specific aims will provide valuable insights for satellite cell-based cell therapies in muscular dystrophies and other muscle diseases. Eventually, our experimental scheme will be directly applied to human satellite cells isolated from DMD patients. The DMD-specific satellite cells stably transfected with CD24 and microdystrophin will be able to produce enough satellite cells for autologous and systemic cell transplantation to DMD patients.

摘要 杜氏肌营养不良症（DMD）是由编码肌营养不良蛋白的基因突变引起的， 保持肌肉纤维结构和功能在整个身体，防止它被损坏， 肌肉收缩。目前，对于DMD患者没有确定的治疗，并且当前的治疗集中于 延长生存期和提高生活质量。持续治疗需要功能性肌营养不良蛋白 在所有受影响的肌肉群中恢复。恢复肌纤维中抗肌萎缩蛋白表达的可能途径 包括细胞疗法，我们希望移植一种能够再生肌肉特定类型的细胞， 帮助我们开发治疗这种疾病的新方法。肌肉干细胞，称为卫星细胞，分离 来自健康供体或患者的细胞应该能够提供肌营养不良蛋白并修复DMD患者的肌肉损伤。 对于有效的DMD治疗，维持自我更新能力的卫星细胞也是必要的。然而，在这方面， 卫星细胞是罕见的（所有肌肉细胞核的2-7%），并且通常难以分离。此外，高效生肌 分化和肌源性分化的扩大仍然难以捉摸，必须进一步发展， 以产生有效的基于细胞的治疗慢性肌肉疾病。患者源性诱导多能干细胞 多能干细胞（iPSC）是获得无限数量的肌源性细胞的理想细胞来源， 移植后排斥。然而，注射细胞的全身递送的失败阻碍了实际应用。 应用于患者。我们的长期目标是为细胞治疗开发有效的卫星细胞供应， 找到一种有效的全身输送方法，可以治疗所有受影响的肌肉，并使注射的细胞， 自我更新并达到有效质量。本申请的具体目的：1.以确定可能 通过实时成像和3-羟色胺测定表达CD 24的卫星细胞和毛细血管内皮细胞之间的相互作用。 在我们最近建立的整个肌肉组织清除协议之后进行三维成像。2.发展系统性的交付 用于卫星细胞和hiPSC衍生的肌源性祖细胞与遗传修饰组合的方法， 包括转导外渗因子、CD 24和甘露糖醇， 透化作用3.开发通过细胞外囊泡（EV）系统递送卫星细胞的方法- 介导的CD 24转移。结合基因和蛋白质转导，这种协调一致的方法将有助于 我们制造卫星细胞，可以全身移植到患者体内，以彻底治愈DMD。的 拟议的具体目标将测试系统注射的卫星细胞是否可以输送到受伤的目标 肌肉和有助于肌肉再生以及卫星细胞库。预期的结果 提出的具体目标将为肌营养不良症中基于卫星细胞的细胞疗法提供有价值的见解。 和其他肌肉疾病。最终，我们的实验方案将直接应用于人类卫星细胞 分离自DMD患者。稳定转染CD 24和microdystrophin的DMD特异性卫星细胞 将能够产生足够的卫星细胞，用于DMD患者的自体和全身细胞移植。