Striated Muscle Gene Transcription and Regulatory Gene 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和旨在改善肌肉中表达治疗性蛋白质的调控盒的应用研究。这两种成分都集中在小鼠M-肌酸激酶基因上。MCK是一个信息模型，因为它在骨骼肌和心肌中表达，并且在不同的肌肉和快/慢肌纤维中以不同的水平表达。这些属性加上MCK固有的高表达使其组分对于在肌肉特异性调节盒中的使用具有吸引力。目的I和2继续研究鉴定与四个MCK区域相关的控制元件和转录因子，所述MCK区域包含具有未知功能的高度保守序列基序块。了解这些区域很重要，因为它们参与了发育过程中的肌肉基因激活、纤维类型特异性基因转录以及对生理信号的反应。总体策略包括肌细胞培养测试的转录活性所赋予的保守序列基序，然后通过测定核因子结合候选控制元件和定量蛋白质组学分析的差异富集的核提取物，以确定转录因子候选人。然后使用ChIP分析来证实候选因子与控制元素的关联。通过系统性病毒递送和转基因小鼠实验，进一步表征了MCK基因在成人肌肉中表达和发育过程中的控制元件作用。这些研究的信息应该广泛适用于许多肌肉基因的调控。目标3和4：该项目的应用方面使用来自目标1和2的控制元件和信息，以及来自其他肌肉基因的控制区域，以设计用于基因治疗的最佳调控盒。从这些盒表达的蛋白质可用于治疗肌肉疾病、肌肉老化和损伤问题，以及其中骨骼肌可用于分泌治疗性蛋白质的疾病，例如，激素和凝血因子疾病。这些盒式磁带也可用于许多基础研究目的。Aim-3将优化设计用于在不同横纹肌类型中发挥作用的盒的转录活性，同时还保持高肌肉特异性以防止在免疫系统和其他非肌肉细胞中表达。另一个目标将集中在设计微型调控盒，其与在AAV和其他病毒载体中的有限包装空间内包装大型治疗性cDNA（例如小型和微型肌营养不良蛋白）兼容。Aim-4的目的是允许治疗产品水平通过无害药物的水平进行外部调节。将优化来自Aim-3的酪蛋白酶以表达先前设计的人工转录因子（ATF）的DNA结合和激活结构域。由于ATF活性需要2个结构域的二聚化，并且由于二聚化的量取决于药物浓度，因此可以通过操纵药物剂量来调节与ATF的独特DNA结合位点连接的治疗性cDNA的转录速率。公共卫生相关性。 肌肉基因控制区和相关转录因子的实验分析是至关重要的，以获得在人类生长发育过程中，以及在健康和患病的成人肌肉基因是如何调节的充分理解。另一个价值是，新发现的控制区可用于创建最佳调控盒，用于遗传性肌肉疾病患者和因创伤或老年原因导致的严重肌肉损失患者的基因治疗。改进的调节盒活性减少了患者治疗所需的病毒载体的数量，从而提高了安全性并降低了病毒生产成本。

DESCRIPTION (provided by applicant): This project combines basic research aimed at understanding how muscle genes are regulated with applied research aimed at improving regulatory cassettes for expressing therapeutic proteins in muscle. Both components focus on the mouse M-creatine kinase gene. MCK is an informative model because it is expressed in skeletal and cardiac muscle, and at varying levels in different muscles & fast/slow muscle fibers. These attributes plus MCK's intrinsically high expression make its components attractive for use in muscle- specific regulatory cassettes. Aims I & 2 continue studies that are identifying control elements & transcription factors associated with four MCK regions containing blocks of highly conserved sequence motifs with unknown functions. Understanding these regions is important because they are involved in muscle gene activation during development, fiber type-specific gene transcription, and response to physiological signals. The overall strategy involves muscle cell culture tests of the transcriptional activity conferred by conserved sequence motifs followed by assays for nuclear factor binding to candidate control elements and quantitative proteomic analysis of differentially-enriched nuclear extracts to identify transcription factor candidates. ChIP analysis is then used to corroborate association of candidate factors with the control elements. Further characterization of control element roles in MCK gene expression in adult muscles & during development is accomplished with systemic viral delivery & transgenic mouse experiments. Information from these studies should be broadly applicable to the regulation of many muscle genes. Aims 3 & 4: Applied aspects of the project use control elements & information from Aims 1 & 2, as well as control regions from other muscle genes, to design optimal regulatory cassettes for gene therapy. Proteins expressed from these cassettes could be used for treating muscle diseases, muscle aging & injury problems, and diseases in which skeletal muscle could be used to secrete therapeutic proteins, e.g., hormone & clotting factor diseases. The cassettes can also be used for many basic research purposes. Aim-3 will optimize the transcriptional activity of cassettes designed to function in different striated muscle types, while also maintaining high muscle specificity to prevent expression in immune system and other non-muscle cells. An additional goal will focus on designing miniature regulatory cassettes that are compatible with packaging large therapeutic cDNAs such as mini- & micro-dystrophins within the limited packaging space in AAV & other viral vectors. The purpose of Aim-4 is to allow therapeutic product levels to be externally regulated by the levels of a non-harmful drug. Cassettes from Aim-3 will be optimized for expressing the DNA-binding & activation domains of previously designed artificial transcription factors (ATFs). Since ATF activity requires dimerization of the 2 domains, and since the amount of dimerization depends on drug concentration, the transcription rate of therapeutic cDNAs linked to unique DNA- binding sites for the ATF can then be regulated by manipulating drug dosage. PUBLIC HEALTH RELEVANCE. Experimental analysis of muscle gene control regions and associated transcription factors is critical for obtaining a full understanding of how muscle genes are regulated during human growth and development and in both healthy and diseased adult muscle. An additional value is that newly discovered control regions can be used to create optimal regulatory cassettes for gene therapy treatments of patients with genetic muscle diseases and with major muscle loss through trauma or geriatric causes. Improved regulatory cassette activity decreases the number of viral vectors needed for patient treatment, thereby increasing safety and decreasing viral production costs.