A Novel Xenograft Model of FSHD

 描述(由申请人提供)：面肩肩周型肌营养不良症(FSHD)是第三种最常见的肌营养不良症，全世界每15,000至20,000人中就有1人受到影响。如果FSHD的有效小鼠模型可用，FSHD的开发和测试疗法将大大推进。理想情况下，小鼠版本的FSHD将复制FSHD肌肉的所有特征，保留新鲜活检中发现的形态、生理和基因组差异。由于导致FSHD的病理生理机制尚不清楚，最好是从FSHD组织本身构建这样的模型，而不是通过操纵特定的基因或基因产物来构建，尽管涉及到这些基因或基因产物，但可能不足以用于发病机制。最后，小鼠应该可以进入许多实验室，因此应该有大量的可用的小鼠。我和我的同事们一直在开发这样一个模型。特别是，我们一直在研究异种移植的使用，以创造具有人源化的胫前肌的小鼠，这些肌肉是从FSHD和对照肌源性细胞系发展而来的。在我们的初步研究中，我们使用了实验室中的常规方法，包括对免疫低下(NRG)小鼠的后肢进行X射线照射，并注射心脏毒素来消除小鼠的胫前肌及其再生能力。我们还采用了新的方法，包括通过腓神经对移植物进行定期电刺激，以及由我们的合作者W.E.Wright博士(德克萨斯大学西南分校)、P.Jones博士(加州大学马萨诸塞州医学院)和L.M.昆克尔博士(波士顿儿童医院)提供的独特试剂和方法。我们用一种表达荧光素酶的人肌源性细胞系获得的初步结果，以便于通过发光术进行监测，结果表明，这些细胞 移植到小鼠的TA隔室，在那里它们存活下来形成成熟的肌肉纤维，这种纤维起源于人类，基本上不含小鼠肌核。人体纤维受到神经支配，在刺激腓神经时收缩，产生与人类肌肉相当的比力。由于移植物含有PAX7阳性的人类细胞，与人类肌纤维相邻，它们也可能具有进一步生长的能力。最近对来自FSHD患者的细胞系的数据表明，这些细胞也能有效地植入，形成专门表达Dux4及其两个下游靶点的肌肉。这表明我们的方法保留了肌源性细胞在植入、生长和分化过程中的遗传特征。我们有两个具体目标：(I)优化成熟人类肌肉组织在小鼠体内的形成，作为开发用于治疗测试的FSHD模型的第一步；以及(Ii)将我们优化的方法应用于FSHD患者及其未受影响的亲属的细胞，以开发、表征和评估一种新的FSHD模型在小鼠身上的变异性，以便后续的治疗测试。因此，我们的研究应该在小鼠身上产生一种FSHD肌肉模型，这将是识别和测试治疗FSHD的药物的宝贵试剂。

 DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is the third most common form of muscular dystrophy and affects 1 in 15,000 to 20,000 people worldwide. Developing and testing therapeutics for FSHD would be significantly advanced if a valid mouse model of the disease were available. Ideally, a murine version of FSHD would reproduce all the features of FSHD muscle, retaining the morphological, physiological and genomic differences found in fresh biopsies. As the pathophysiological mechanisms that lead to FSHD are still unclear, it would be best if such a model were constructed from FSHD tissue itself, rather than by manipulating particular genes or gene products that, although involved, may not be sufficient for pathogenesis. Finally, the mice should be accessible to many laboratories and so should be available in significant numbers. My colleagues and I have been developing such a model. In particular, we have been investigating the use of xenografting to create mice with humanized Tibialis anterior muscles developed from FSHD and control myogenic cell lines. In our preliminary studies, we have used methods that are routine in our laboratory, including X-irradiation of the lower hindlimbs of immunocompromised (NRG) mice and injection of cardiotoxin to eliminate the Tibialis anterior muscles of the mice and their abiliy to regenerate. We have also incorporated new methods, including periodic electrical stimulation of the graft through the peroneal nerve, and unique reagents and methods provided by our collaborators, Drs. W.E. Wright (UT Southwestern), P. Jones (UMass Med School) and L.M. Kunkel (Boston Children's Hospital). Our preliminary results, obtained with a human myogenic cell line that expresses luciferase, to facilitate monitoring by luminometry, show that these cells engraft into the otherwise empty TA compartment of mice, where they survive to form mature muscle fibers that are human in origin and that are essentially free of murine myonuclei. The human fibers are innervated and they contract upon stimulation of the peroneal nerve, to generate a specific force comparable to that of human muscle. As the grafts contain PAX7-positive human cells located adjacent to human myofibers, they are also likely to have the capacity for further growth. Recent data with a cell line derived from an FSHD patient indicate that these cells engraft efficiently as well, forming muscles that specifically express Dux4 and two of its downstream targets. This suggests that our methods preserve the genetic characteristics of myogenic cells as they engraft, grow, and differentiate. We have two specific aims: (i) to optimize the formation of mature human muscle tissue in mice, as a first step in developing a model of FSHD for therapeutic testing; and (ii) to apply our optimized methods to cells from FSHD patients and their unaffected relatives, to develop, characterize, and assess the variability of a novel model of FSHD in mice, for subsequent therapeutic testing. Our studies should therefore generate a model of FSHD muscle in mice that will be an invaluable reagent in identifying and testing drugs to treat FSHD.