EOM satellite cells for DMD therapy

 描述(申请人提供)：Duchenne肌营养不良症(DMD)是一种严重的肌肉萎缩疾病，与功能性肌营养不良蛋白的丧失有关，肌营养不良蛋白是一种对肌膜稳定至关重要的蛋白质。在人类和动物模型中，眼外肌(EOMS)是唯一不受dystrophin缺乏影响的组织。EOM卫星细胞(SCs，成体成肌祖细胞)的特异性可能在EOM选择性备用中起一定作用。这一探索性项目旨在评估EOM SCs作为基于细胞的疗法对抗DMD的有效来源的潜力。我们的小鼠研究表明，新鲜分离的EOM与肢体干细胞相比，在植入潜力和体外扩张能力方面具有优势。令人印象深刻的是，EOM SCs建立了大量的更新细胞，在重新培养时仍保持旺盛的扩张能力。因此，使用EOM SCs作为供体成肌细胞的来源，可以绕过肢体SCs有限的扩增能力，满足临床上对大量祖细胞的需求。值得注意的是，最终可以开发出一种用于人类EOM检索的协议，其方式类似于当前的眼库操作。然而，重要的是首先确定EOM SCs或其后代是否会在Dstrophin缺乏影响的宿主环境中继续保持其高性能能力。这项拟议的研究还将提供有关新生幼崽肌源性细胞植入结果的关键数据，与较“传统”的老年动物植入范例相比。随着新生儿DMD的筛查进入临床，在出生后不久确定成肌细胞替代的方法是重要的。在拟议的研究中，从成年小鼠的EOMS中分离出的供体干细胞将被移植到dystrophin缺陷(Mdx4cv)宿主小鼠的肌肉中。移植结果将通过监测供者衍生的报告表达、肌萎缩蛋白在肌膜中的恢复、中央肌核的范围和肌肉性能来决定。因此，这项应用的具体目的是：1.研究供体EOM SCs肌肉注射到新生(3日龄)和新断奶(24日龄)宿主MDX小鼠后的植入结果。重点放在短期(6-12周龄)和长期(24-48周龄)植入结果。2.改进体外扩增EOM SC后代的方法，并研究扩增后的细胞在体内的植入结果(如目标1所示)。人们认识到，虽然MDX小鼠已被广泛用作DMD的实验室模型，但MDX的病理远没有DMD患者严重。尽管如此，对MDX小鼠的研究已经为随后的营养不良狗研究和人类临床试验提供了重要的指导数据。我相信 本文所考虑的方法将成功地为对抗DMD和其他肌肉萎缩情况的新的临床方法提供新的见解。

 DESCRIPTION (provided by applicant): Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease associated with the loss of functional dystrophin, a protein essential for sarcolemmal stability. Extraocular muscles (EOMs) are uniquely unaffected by dystrophin deficiency in humans and in animal models. Specific traits of EOM satellite cells (SCs, adult myogenic progenitors) may play a role in EOM preferential sparing. This exploratory project aims to evaluate the potential of EOM SCs as effective source for cell-based therapy to combat DMD. Our mouse studies showed superiority of freshly isolated EOM vs. limb SCs, in engraftment potential and ex-vivo expansion capability. Impressively, EOM SCs established numerous renewal cells that maintain vigorous expansion ability upon re-culturing. Hence, the use of EOM SCs as a source for donor myoblasts could allow bypassing the limited expansion capacity encountered with limb SCs, and meeting the demand for a high number of progenitors essential for clinical applications. Significantly, a protocol can be developed eventually for human EOM retrieval in a manner similar to current Eye Bank operations. It is important however to determine first if EOM SCs or their progeny would continue to maintain their high-performance capacity in dystrophin-deficiency affected (limb) host environment. The proposed studies will also provide key data about the outcome of myogenic cell engraftment to newly born pups compared with the more "traditional" engraftment paradigm to older animals. As newborn screening of DMD is entering the clinics, identifying means for myoblast replacement soon after birth is of importance. In the proposed studies, donor SCs isolated from EOMs of adult mice will be transplanted into muscles of dystrophin-deficient (mdx4cv) host mice. Transplantation outcome will be determined by monitoring donor-derived reporter expression, restoration of dystrophin in the sarcolemma, extent of central myonuclei and muscle performance. Accordingly, the specific aims of this application are: 1. Investigate engraftment outcome following intra- muscular delivery of donor EOM SCs injected to newly born (3-day-old) and newly weaned (24-day-old) host mdx mice. Emphasis is given to short-term (at 6 & 12 weeks of age) and long-term (at 24 & 48 weeks of age) engraftment outcomes. 2. Refine means for ex-vivo expansion of EOM SC progeny and investigate in- vivo engraftment outcomes with the expanded cells (as in Aim 1). It is recognized that while the mdx mouse has been used extensively as a laboratory model of DMD, the mdx pathology is far less severe than that of DMD patients. Still, studies with mdx mice have generated important directive data for subsequent studies with dystrophic dogs and for pilot clinical trials in human. I am confident that the approaches considered herein will be successful in contributing new insights toward novel clinical approaches for combating DMD and other muscle wasting conditions.