Targeting Dystroglycanopathies using Pluripotent-derived Myogenic Progenitors

总结 肌营养不良症是与以下疾病相关的肌营养不良症的常见且异质子集： α-肌营养不良聚糖 (α-DG) 的异常 O-糖基化。它们通常是由 fukutin 突变引起的 相关蛋白（FKRP），编码一种假定的基于高尔基体的糖基转移酶，从而产生广泛的 MD表型谱；大多数病例被归类为先天性 MD 1 型 (MDC1C) 或肢带 MD 2I 型 (LGMD2I)。就 LGMD2I 而言，临床表现从严重的早发型到轻度的晚发型不等。 发病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) 的这一关键成分功能障碍，并且 从而导致细胞膜损伤和肌肉细胞凋亡，导致慢性组织损伤 退化和肌肉收缩力受损。虽然目前对此还没有有效的治疗方法 或任何其他类型的肌营养不良症，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增殖，同时保持 分化潜能，它们代表了治疗应用的有利选择。如果是 肌营养不良症，无论是同种异体还是自体细胞移植都有可能导致 有效的治疗。对于同种异体移植，可以利用 iPS 衍生的肌源性祖细胞 从健康的 HLA 匹配供体获得，移植后将产生新的健康 肌纤维。自体方法需要在使用之前对营养不良的 iPS 细胞进行离体遗传校正。 移植。我们的团队开创了从小鼠和人类中提取骨骼肌原细胞的方法 在早期中胚层发育过程中通过短暂诱导 Pax3 或 Pax7 形成多能 ES /iPS 细胞。 将这些细胞移植到杜氏肌营养不良症 (DMD) 小鼠模型中，可产生肌纤维 以及卫星细胞植入，伴随着肌肉力量产生的改善。 在这里，我们建议首次研究基于多能干细胞的肌肉治疗方法 与 FKRP 突变相关的疾病。使用 LGMD2I 小鼠模型得出的令人兴奋的初步数据揭示了 小鼠多能源性肌源性祖细胞移植严重受影响的隔膜的能力 全身给药后的小鼠。在目标 1 中，我们将更详细地研究隔膜植入。在目标 2 中，我们 提议建立基因编辑工具来纠正患者特异性 iPS 细胞的 FKRP 突变 CRISPR/Cas9系统，将在体外和体内进行验证。最后，开始打基础 用于肌肉疾病的多能干细胞的临床开发，目标 3 将重点关注可扩展性， 多能源性肌原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.