Muscle Gene Regulation and Cassettes for Gene Therapy

描述(由申请人提供)：该项目结合了一个基础研究部分，旨在了解肌肉基因是如何调控的，以及一个应用部分，旨在设计用于在横纹肌中表达治疗性蛋白质的调节盒。以往的基础研究主要集中在了解小鼠肌酸激酶(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定位另外三个赋予增强子-启动子复合体更高转录活性的调节区，以及第四个赋予基因拷贝数依赖表达的区域。这些区域内的控制元件将被识别，然后这些序列将被用来识别和理解它们相关的转录因子的功能。该项目的应用方面利用上述基本信息以及已发表的关于其他横纹肌基因的数据来构建调节盒，这些调节盒将用于治疗骨骼肌和心肌疾病，以及可能受益于将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不同类型横纹肌的转录活性，同时保持紧密的肌肉特异性转录，以防止免疫系统和其他非肌肉细胞中的治疗性基因表达，这些基因可能被错误靶向的基因治疗载体无意中转导和破坏。另外两个目标是构建微型调节盒，与将微型和微肌营养不良蛋白等治疗性cDNA包装到AAV和其他小型病毒载体中兼容，以及构建高活性的肌肉特异性转录因子，表达外部可调节的转录因子，对无害的药物做出选择性转录，从而允许治疗基因产品水平的外部操作。

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.