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。这两个成分都集中在小鼠m -肌酸激酶基因上。MCK是一个信息丰富的模型，因为它在骨骼肌和心肌中表达，并且在不同的肌肉和快/慢肌纤维中表达的水平不同。这些特性加上MCK本质上的高表达使其成分在肌肉特异性调节盒中具有吸引力。目标1和目标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，这些区域含有功能未知的高度保守序列基序块。了解这些区域很重要，因为它们参与发育过程中的肌肉基因激活、纤维类型特异性基因转录和对生理信号的反应。总体策略包括肌肉细胞培养测试保守序列基序所赋予的转录活性，然后是核因子与候选控制元件结合的测定和差异富集核提取物的定量蛋白质组学分析，以确定转录因子候选。然后使用ChIP分析来证实候选因素与控制因素的关联。通过系统病毒传递和转基因小鼠实验，进一步表征了控制元件在成年肌肉和发育过程中MCK基因表达中的作用。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目标1和2的控制元素和信息，以及其他肌肉基因的控制区，来设计基因治疗的最佳调节盒。从这些磁带中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。盒式磁带还可用于许多基础研究目的。Aim-3将优化在不同横纹肌类型中发挥作用的卡带的转录活性，同时保持高肌肉特异性，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节盒，与在AAV和其他病毒载体的有限包装空间内包装大型治疗性cdna（如迷你和微型营养不良蛋白）兼容。Aim-4的目的是允许治疗产品水平受非有害药物水平的外部调节。Aim-3的卡带将被优化，用于表达先前设计的人工转录因子（atf）的dna结合和激活域。由于ATF活性需要这两个结构域的二聚化，并且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独特DNA结合位点相连的治疗性cdna的转录率。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对于全面了解在人类生长发育过程中以及在健康和患病的成人肌肉中肌肉基因是如何被调节的至关重要。另一个价值是，新发现的控制区可用于为遗传性肌肉疾病患者和因创伤或老年性原因导致的主要肌肉损失患者的基因治疗创造最佳的调节磁带。改进的监管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.