A Novel Xenograft Model of FSHD

一种新型的 FSHD 异种移植模型

• 批准号: 8930205
• 负责人: ROBERT J BLOCH
• 金额: $ 39.75万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-30 至 2017-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8930205
• 关键词: Affect; Biological Markers; Biopsy; Boston; Cell Line; Cells; Characteristics; Chromosomes; Communities; Contracts; D4Z4; Data; Development; Direct Lytic Factors; Disease; DsRed; Electric Stimulation; Engraftment; Exhibits; Facioscapulohumeral Muscular Dystrophy; Family; Family member; Fiber; Fluorescence; Genes; Genetic; Genetic screening method; Genomics; Goals; Growth; Hindlimb; Human; Immunocompromised Host; In Vitro; Individual; Injection of therapeutic agent; Label; Laboratories; Lead; Left; Link; Luciferases; Measures; Methods; Modeling; Molecular; Monitor; Mus; Muscle; Muscle Fibers; Muscular Dystrophies; Natural regeneration; Neuromuscular Junction; PAX7 gene; Pathogenesis; Patients; Pediatric Hospitals; Pharmacy (field); Physiological; Procedures; Production; Property; Proteins; Rag1 Mouse; Reagent; Relative (related person); Research; Schools; Scientist; Source; Staining method; Stains; Testing; Therapeutic; Tissues; Torque; Xenograft Model; Xenograft procedure; cohort; drug testing; improved; irradiation; man; mouse model; muscle form; muscle regeneration; neuromuscular stimulation; novel; peroneal nerve; polyadenylated messenger RNA; precursor cell; public health relevance; tibialis anterior muscle; tool

 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.

 描述（由申请人提供）：面肩肱型肌营养不良症（FSHD）是第三种最常见的肌营养不良症，影响全球15，000至20，000人中的1人。如果有一个有效的FSHD小鼠模型，那么FSHD治疗方法的开发和测试将大大推进。理想情况下，FSHD的小鼠版本将再现FSHD肌肉的所有特征，保留新鲜活检中发现的形态学，生理学和基因组差异。由于导致FSHD的病理生理机制尚不清楚，因此最好从FSHD组织本身构建这种模型，而不是通过操纵特定的基因或基因产物，尽管涉及，但可能不足以致病。最后，许多实验室都应该能够获得小鼠，因此应该有大量的小鼠。我和我的同事一直在开发这样一个模型。特别是，我们一直在研究使用异种移植来创建具有从FSHD和对照肌源性细胞系开发的人源化胫骨前肌的小鼠。在我们的初步研究中，我们使用了我们实验室常规的方法，包括X射线照射免疫受损（NRG）小鼠的下肢和注射心脏毒素以消除小鼠的胫骨前肌及其再生能力。我们还采用了新的方法，包括通过腓神经对移植物进行周期性电刺激，以及我们的合作者W.E.赖特（UT西南），P.琼斯（马萨诸塞大学医学院）和L.M.波士顿儿童医院（Boston Children's Hospital）我们的初步结果，获得了与人类肌细胞系，表达荧光素酶，以促进监测光度计，表明这些细胞， 移植到小鼠的空TA隔室中，在那里它们存活以形成成熟的肌纤维，所述肌纤维是人源的并且基本上不含鼠肌核。人体纤维受神经支配，并且它们在腓神经刺激时收缩，以产生与人体肌肉相当的特定力。由于移植物含有位于人类肌纤维附近的PAX 7阳性人类细胞，因此它们也可能具有进一步生长的能力。来自FSHD患者的细胞系的最新数据表明，这些细胞也有效地移植，形成特异性表达Dux 4及其两个下游靶点的肌肉。这表明我们的方法在肌源性细胞移植、生长和分化时保留了肌源性细胞的遗传特征。我们有两个具体目标：（i）优化小鼠中成熟人类肌肉组织的形成，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来自FSHD患者及其未受影响的亲属的细胞，以开发、表征和评估小鼠中FSHD的新模型的可变性，用于随后的治疗测试。因此，我们的研究应该在小鼠中产生FSHD肌肉模型，这将是鉴定和测试治疗FSHD的药物的宝贵试剂。