Targeting Dystroglycanopathies using Pluripotent-derived Myogenic Progenitors

总结 肌营养不良是一种常见的和异质性的肌营养不良亚类， α-肌营养不良蛋白聚糖（α-DG）的异常O-糖基化。它们通常是由基因突变引起的， 相关蛋白（FKRP），其编码假定的基于高尔基体的糖基转移酶，导致广泛的 MD表型谱;大多数病例被归类为先天性MD 1型（MDC 1C）或肢带型 MD 2I型（LGMD 2I）。在LGMD 2 I的情况下，临床表现从严重的早发性到轻度的迟发性不等。 开始MD。FKRP相关疾病的生化标志是α-DG的低糖基化， 导致肌营养不良蛋白糖蛋白复合物（DGC）的这一关键组分的功能障碍，以及 从而导致细胞膜损伤和肌肉细胞凋亡，导致慢性组织损伤。 退化和受损的肌肉收缩力。虽然目前没有有效的治疗方法， 或任何其他类型的营养不良聚糖病，一种有吸引力的治疗方法是使用基于细胞的疗法， 促进肌肉再生。因为多能干细胞可以无限扩增， 由于具有分化潜能，它们代表了治疗应用的有利选择。的情况下 肌营养不良症，无论是同种异体或自体细胞移植都有可能导致 有效治疗。对于同种异体移植，将利用iPS衍生的肌源性祖细胞 从健康的HLA匹配的供体获得，其在移植后将产生新的健康的 肌纤维自体方法将需要营养不良的iPS细胞的离体遗传校正，然后再将其移植到细胞。 移植我们的团队开创了从小鼠和人类中获得骨骼肌细胞的方法 在早期中胚层发育期间，通过Pax 3或Pax 7的瞬时诱导来诱导多能ES /iPS细胞。 将这些细胞移植到杜氏肌营养不良症（DMD）的小鼠模型中， 以及伴随着肌肉力量产生的改善的卫星细胞移植。 在这里，我们提出了第一次调查的多能干细胞为基础的治疗方法，肌肉 与FKRP突变相关的疾病。令人兴奋的初步数据，使用LGMD 2 I小鼠模型，揭示了 小鼠多能性来源的肌源性祖细胞移植严重受影响的膈肌的能力， 小鼠全身给药。在目标1中，我们将进一步详细研究横膈膜植入。在目标2中， 我们建议建立基因编辑工具，以纠正患者特异性iPS细胞的FKRP突变， CRISPR/Cas9系统，将在体外和体内进行验证。最后，开始为 用于肌肉疾病的多能干细胞的临床开发，目标3将集中在可扩展性上， 多能性衍生的肌原性祖细胞的纯化和安全性。

Summary Dystroglycanopathies are a common and heterogeneous subset of muscular dystrophies associated with abnormal O-glycosylation of alpha-dystroglycan (α-DG). They are frequently caused by mutations in fukutin- related protein (FKRP), which encodes a putative Golgi-based glycosyltransferase, resulting in a broad spectrum of MD phenotypes; with most cases being classified as congenital MD type 1 (MDC1C) or limb-girdle MD type 2I (LGMD2I). In the case of LGMD2I, clinical presentation varies from severe early-onset to mild late- onset MD. The biochemical hallmark of FKRP-associated diseases is the hypoglycosylation of α-DG, which results in dysfunction of this critical component of the dystrophin glycoprotein complex (DGC), and consequently leads to cell membrane damage and apoptosis of muscle cells, resulting in chronic tissue degeneration and impaired muscle contractility. Although no effective treatment is available at present for this or any other type of Dystroglycanopathy, one attractive therapeutic approach is to use cell based therapies to promote muscle regeneration. Because pluripotent stem cells can be expanded indefinitely, while maintaining differentiation potential, they represent an advantageous option for therapeutic application. In the case of muscular dystrophies, either allogeneic or autologous cell transplantations have the potential to lead to an effective treatment. For allogeneic transplantation, one would utilize iPS-derived myogenic progenitors obtained from a healthy HLA-matched donor, which following transplantation would give rise to new healthy myofibers. The autologous approach would require ex vivo genetic correction of dystrophic iPS cells prior to transplantation. Our group has pioneered methods to derive skeletal myogenic cells from mouse and human pluripotent ES /iPS cells through transient induction of Pax3 or Pax7 during early mesoderm development. Transplantation of these cells in mouse models for Duchenne Muscular Dystrophy (DMD), results in myofiber and satellite cell engraftment that is accompanied by improvement in muscle force generation. Here we propose to investigate for the first time a pluripotent stem cell-based therapy approach for muscle diseases associated with FKRP mutations. Exciting preliminary data, using a LGMD2I mouse model, reveal the ability of mouse pluripotent-derived myogenic progenitors to engraft the severely affected diaphragm of these mice upon systemic delivery. In Aim 1, we will investigate diaphragm engraftment in further detail. In Aim 2, we propose to establish gene editing tools to correct FKRP mutations of patient-specific iPS cells using the CRISPR/Cas9 system, which will be validated in vitro and in vivo. Finally, to begin laying the groundwork for clinical development of pluripotent stem cells for muscle diseases, in Aim 3 will focus on the scalability, purification, and safety of pluripotent-derived myogenic progenitors.