Dysferlin: Approaches to Domain Analysis and Gene Therapy

膜修复是一个基本的细胞生存过程中的大型，不可替代的，或经常受伤的细胞类型 比如肌肉细胞。我们曾报道，骨骼肌蛋白dysferlin是有缺陷的三好 肌病（MM）和肢带型肌营养不良2B型（LGMD2B），以及dysferlin加速肌肉 膜修复通过钙触发机制，调用结合膜联蛋白和形成 胞内囊泡聚集体。因此，MM和LGMD2B似乎可能是异常的 骨骼肌膜修复我们现在建议定义dysferlin的结构域，这些结构域对此至关重要。 修复过程，并确定截短形式的"mini-dysferlin"是否可以拯救MM/LGMD2B 表型。我们的研究有五个具体的目的：（1）产生和表征转基因小鼠， 表达全长和截短的dysferlin。假设：表达超正常水平的全长或 截短的dysferlin可以挽救dysferlin缺陷模型中的营养不良表型。(2)表征 截短的dysferlin的生物化学性质。假设：对全长和截短的 dysferlin蛋白将鉴定保留全长蛋白功能特性的结构域。（三） 通过mini-dysferlin蛋白表征膜修复。假设：微dysferlin蛋白保留 功能域将增强dysferlin缺陷肌细胞中的膜修复。(4)颅内分娩 候选AAV8包装的微型dysferlin基因。假设：全身递送迷你dysferlin基因将 引起这些基因在肌肉中的传播并增强膜修复。(5)分析斑马鱼 dysferlin的存在和dysferlin同源物下调时的表型。 假设：Dysferlin缺乏将在斑马鱼中产生肌病表型，并且有可能 分析替代斑马鱼中部分或全部形式的dysferlin的影响。方法：我们将使用 各种技术来定义dysferlin的功能结构域，然后将分析对 通过转基因体内递送至肌肉的全长和截短的dysferlin的dysferlin缺陷表型 小鼠和全身施用AAV8。意义：本研究将（1）加深对 骨骼肌肌细胞膜修复的分子生物学;（2）提供对骨骼肌细胞膜修复的深入了解。 dysferlin的功能结构域;（3）确定开发用于拯救 由dysferlin缺乏引起的肌肉表型;以及（4）开发关于 全身给予MM和LGMD2B的病毒基因治疗。

Membrane repair is a fundamental cell survival process in large, irreplaceable, or frequently injured cell types such as muscle cells. We have reported that the skeletal muscle protein dysferlin is defective in Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and that dysferlin accelerates muscle membrane repair via a calcium-triggered mechanism that invokes binding to annexins and the formation of intracellar vesicular aggregates. It thus seems likely that MM and LGMD2B are a consequence of aberrant skeletal muscle membrane repair. We now propose to define domains of dysferlin that are critical for this repair process and determine whether truncated forms of "mini-dysferlin" can rescue the MM/LGMD2B phenotype. Our study has five Specific Aims: (1) Generate and characterize transgenic mice that over- express full-length and truncated dysferlin. Hypothesis: expression of supra-normal levels of full-length or truncated dysferlin can rescue the dystrophic phenotype in a dysferlin-deficient model. (2) Characterize the biochemical properties of truncated dysferlin. Hypothesis: A biochemical analysis of full-length and truncated dysferlin proteins will identify domains retaining functional properties of the full-length protein. (3) Characterize membrane repair by mini-dysferlin proteins. Hypothesis: mini-dysferlin proteins retaining functional domains will augment membrane repair in dysferlin-deficient muscle cells. (4) Intravenously deliver candidate AAV8-packaged mini-dysferlin genes. Hypothesis: systemic delivery of mini-dysferlin genes will give rise to dissemination of these genes in muscle and enhanced membrane repair. (5) Analyze zebrafish for the presence of dysferlin and for phenotypes when dysferlin homologues are down-regulated. Hypothesis: Dysferlin-deficiency will produce a myopathic phenotype in zebrafish and it will be possible to analyze the impact of replacement of partial or full forms of dysferlin in the zebrafish. Methods: We will use a variety of techniques to define functional domains of dysferlin and will then analyze the rescue of the dysferlin-deficient phenotypes by full length and truncated dysferlin delivered to muscle in vivo via transgenic mice and systemically administered AAV8. Significance: This study willl (1) enhance our understanding of the molecular biology of sarcolemmal membrane repair in skeletal muscle; (2) provide insight into the functional domains of dysferlin; (3) determine the feasibility of developing mini-dysferlins for rescue of muscle phenotypes resulting from dysferlin deficiency; and (4) develop pilot data on the efficacy of systemically administered viral gene therapy for MM and LGMD2B.