EOM satellite cells for DMD therapy

 描述（由申请人提供）：杜氏肌营养不良症（DMD）是一种严重的肌肉萎缩性疾病，与功能性肌营养不良蛋白（一种肌膜稳定性所必需的蛋白质）的丧失有关。在人类和动物模型中，眼外肌（EOM）是唯一不受肌营养不良蛋白缺乏影响的。EOM卫星细胞（SC，成人肌源性祖细胞）的特定特征可能在EOM优先保留中发挥作用。该探索性项目旨在评估EOM SC作为基于细胞的治疗以对抗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SC相比，在植入潜力和离体扩增能力方面具有优势。令人印象深刻的是，EOM SC建立了许多更新细胞，这些细胞在再培养时保持旺盛的扩增能力。因此，使用EOM SC作为供体成肌细胞的来源可以允许绕过肢体SC遇到的有限扩增能力，并满足临床应用所必需的大量祖细胞的需求。值得注意的是，最终可以开发一种协议，以类似于当前眼库操作的方式用于人类EOM检索。然而，重要的是首先确定EOM SC或其后代是否会在肌营养不良蛋白缺陷影响的（肢体）宿主环境中继续保持其高性能能力。拟议的研究还将提供有关肌源性细胞移植到新生幼崽的结果与更“传统”的移植到老年动物的结果相比的关键数据。随着DMD的新生儿筛查进入临床，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，从成年小鼠的眼外肌分离的供体SC将被移植到肌营养不良蛋白缺陷（mdx4cv）宿主小鼠的肌肉中。移植结果将通过监测供体来源的报告基因表达、肌膜中肌营养不良蛋白的恢复、中央肌核的范围和肌肉性能来确定。因此，本申请的具体目的是：1.研究肌内递送供体EOM SC后的植入结果，所述供体EOM SC注射至新生（3天龄）和新断奶（24天龄）的宿主mdx小鼠。重点是短期（6和12周龄）和长期（24和48周龄）植入结果。2.优化EOM SC子代的离体扩增方法，并研究扩增细胞的体内植入结果（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病理学远不如DMD患者严重。尽管如此，对mdx小鼠的研究为后续的营养不良犬的研究和人类的初步临床试验提供了重要的指导数据。我深信 本文所考虑的方法将成功地为对抗DMD和其它肌肉消耗状况的新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.