Systemic delivery of muscle stem cell for muscle disease therapy

摘要 Duchenne肌营养不良症(DMD)是由肌营养不良蛋白基因突变引起的，该基因的功能 保持全身肌肉纤维的结构和功能，防止肌肉纤维在运动过程中受损 肌肉收缩。目前，对于DMD患者还没有明确的治疗方法，目前的治疗方法主要集中在 延长生存时间，提高生活质量。最终的治疗将需要功能性肌营养不良蛋白 在所有受影响的肌肉群中都恢复了。恢复肌纤维中肌营养不良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的特定类型的细胞将 帮助我们开发治疗这种疾病的新方法。分离的肌肉干细胞，称为卫星细胞 来自健康捐赠者或患者的应该能够提供抗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， 卫星细胞很少见(占所有肌肉细胞核的2-7%)，而且通常很难分离。此外，高效的生肌 分化和扩大肌源性分化仍然难以捉摸，必须按顺序进一步发展。 为慢性肌肉疾病产生有效的基于细胞的疗法。患者来源的诱导多能干细胞 细胞(Ipscs)是获得不限数量的逃避免疫的肌源性细胞的理想细胞来源。 移植后的排斥反应。然而，注射细胞系统性输送的失败阻碍了实际应用。 在患者中的应用。我们的长期目标是发展有效的卫星细胞供应，用于细胞治疗， 找到一种有效的全身给药方法，可以治疗所有受影响的肌肉，并使注射的细胞能够 自我更新，达到有效质量。本申请的具体目的：1.确定可能的 表达CD24的卫星细胞与毛细血管内皮细胞相互作用的实时成像和3-甲基-4-氨基丁酸 在我们最近建立的全肌肉组织清除方案之后，进行了3D成像。2.发展系统性投放 卫星细胞和hPSC衍生的肌祖细胞与遗传修饰相结合的方法， 包括外渗因子CD24和甘露醇的转导，甘露醇是一种增强血管的药剂 渗透作用。3.通过细胞外小泡(EV)开发卫星细胞的系统递送方法- 介导的CD24转移。与基因和蛋白质转导相结合，这种协调一致的方法将有助于 美国将制造可系统移植到患者体内的卫星细胞，以彻底治愈DMD。这个 拟议的特定目标将测试系统注射的卫星细胞是否可以运送到受伤的目标 肌肉和有助于肌肉再生以及卫星细胞库。这次会议的预期结果 提出的具体目标将为肌营养不良症的卫星细胞细胞治疗提供有价值的见解 以及其他肌肉疾病。最终，我们的实验计划将直接应用于人类卫星细胞。 分离自DMD患者。稳定表达CD24和微肌营养不良蛋白的DMD特异性卫星细胞 将能够产生足够的卫星细胞，用于DMD患者的自体和系统细胞移植。

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.