Muscle Gene Regulation and Cassettes for Gene Therapy

描述（由申请人提供）：该项目结合了旨在了解肌肉基因如何调控的基础研究部分，以及旨在设计用于在横纹肌中表达治疗性蛋白质的调控盒的应用部分。 以前的基础研究主要集中在了解小鼠M-肌酸激酶（MCK）基因增强子和启动子如何控制MCK在骨骼肌和心肌中的表达。 这些研究将继续进行，但我们未来的大部分工作将致力于绘制三个额外的调控区域，赋予增强子-启动子复合物更高的转录活性，以及赋予基因拷贝数依赖性表达的第四个区域。 将鉴定这些区域内的控制元件，然后将这些序列用于鉴定和理解其相关转录因子的功能。该项目的应用方面利用上述基本信息，以及有关其他横纹肌基因的已发表数据，构建可用于治疗骨骼肌和心肌疾病的调控盒，以及可受益于使用骨骼肌作为分泌蛋白质来源的治疗情况;例如，激素和凝血因子缺乏症以及组织愈合。 这些研究的目标是优化设计用于在不同横纹肌类型中起作用的盒的转录活性，同时保持紧密的肌肉特异性转录，以防止治疗性基因在免疫系统和其他非肌肉细胞中表达，这些细胞可能被错误靶向的基因治疗载体无意中转导和损伤。 两个额外的目标是构建微型调节盒，其将与将治疗性cDNA（如微型和微小肌营养不良蛋白）包装到AAV和其他小病毒载体中相容，并构建表达外部可调节转录因子的高活性肌肉特异性盒，所述转录因子将响应于无害药物选择性地转录治疗性cDNA，从而允许治疗性基因产物水平的外部操纵。

DESCRIPTION (provided by applicant): This project combines a basic research component aimed at understanding how muscle genes are regulated, with an applied component aimed at designing regulatory cassettes for expressing therapeutic proteins in striated muscle. Prior basic studies concentrated primarily on understanding how the mouse M-creatine kinase (MCK) gene enhancer and promoter control MCK expression in skeletal and cardiac muscle. These studies will continue but much of our future effort will be directed toward mapping three additional regulatory regions which confer higher transcriptional activity to the enhancer-promoter complex, and a fourth region which confers gene copy number-dependent expression. Control elements within these regions will be identified and these sequences will then be used to identify and understand the function of their associated transcription factors. Applied aspects of the project utilize the basic information above, together with published data concerning other striated muscle genes, to construct regulatory cassettes that will be useful in treating skeletal and cardiac muscle diseases, and in therapeutic situations which could benefit from using skeletal muscle as a source of secreted proteins; e.g., hormone and clotting factor deficiency diseases, and tissue healing. Goals for these studies are to optimize the transcriptional activity of cassettes designed to function in different striated muscle types, while simultaneously maintaining tight muscle-specific transcription so as to prevent therapeutic gene expression in immune system and other non-muscle cells that may be inadvertently transduced and damaged by mis-targeted gene therapy vectors. Two additional goals are to build miniature regulatory cassettes that will be compatible with packaging therapeutic cDNAs such as mini- and micro-dystrophins into AAV and other small viral vectors, and to build high activity muscle-specific cassettes expressing externally regulatable transcription factors that will selectively transcribe therapeutic cDNAs in response to non-harmful drugs, thus permitting the external manipulation of therapeutic gene product levels.