Targeting Dystroglycanopathies using Pluripotent-derived Myogenic Progenitors

摘要 肌营养不良症是一种常见的、不同种类的肌营养不良症，与 α-营养不良多糖O-糖基化异常(α-DG)。它们通常是由Fukutin的突变引起的- 相关蛋白(FKRP)，它编码一个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(MDC1C)或四肢带状 MD2I型(LGMD2I型)。在LGMD2I的病例中，临床表现从严重的早发到轻度的迟发- 首发MD。FKRP相关疾病的生化标志是α-DG的低糖基化，这是 导致营养不良蛋白糖蛋白复合体(DGC)的这一关键成分功能障碍，以及 从而导致细胞膜的破坏和肌肉细胞的凋亡，导致组织的慢性 退化和肌肉收缩能力受损。虽然目前还没有有效的治疗方法 或任何其他类型的糖营养不良症，一个有吸引力的治疗方法是使用基于细胞的疗法来 促进肌肉再生。因为多能干细胞可以无限扩增，同时保持 它们具有分化潜能，是治疗应用的有利选择。在.的情况下 肌营养不良症，无论是同种或自体细胞移植，都有可能导致 有效的治疗。对于同种异体移植，人们将利用iPS来源的肌源性祖细胞 取自健康的人类白细胞抗原相合的供者，移植后将产生新的健康 肌纤维。自体方法需要对营养不良的iPS细胞进行体外遗传纠正，然后才能 移植。我们团队已率先从小鼠和人类中分离出骨骼肌源性细胞。 在早期中胚层发育过程中瞬时诱导Pax3或Pax7的多能性ES/iPS细胞。 将这些细胞移植到Duchenne肌营养不良症(DMD)小鼠模型中，产生肌纤维 以及伴随着肌肉力量生成的改善的卫星细胞植入。 在这里，我们建议首次研究一种基于多能干细胞的肌肉治疗方法。 与FKRP突变相关的疾病。令人兴奋的初步数据，使用LGMD2I小鼠模型，揭示了 小鼠多能来源的肌源性祖细胞移植严重受损的横隔膜的能力 小鼠全身给药。在目标1中，我们将更详细地研究横隔膜植入术。在目标2中，我们 建议建立基因编辑工具来纠正患者特有的iPS细胞的FKRP突变 CRISPR/Cas9系统，将在体外和体内进行验证。最后，要开始为 肌肉疾病多能干细胞的临床开发，目标3将重点放在可伸缩性上， 多能来源的肌源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.