Structure of the dystrophin rod in relation to exon boundaries and exon skipping

描述（由申请人提供）：本项目的目的是了解肌营养不良蛋白杆区域的性质。虽然该区域构成了肌营养不良蛋白序列的大部分，但小得多的末端球状结构域通常被认为更重要，因为介导肌营养不良蛋白与其他细胞组分的相互作用。在许多情况下，正在研究缩短版本的肌营养不良蛋白，其中部分杆区域被去除-所谓的迷你肌营养不良蛋白-作为可能的基因治疗替代品。然而，在动物模型中已经观察到大量的背景效应，其中涉及不同杆修饰的略微不同构建的微型肌养蛋白具有显著不同的功效。同样，最近已经证明，肌营养不良蛋白基因的加工通过外显子跳读天然产生各种缺失（至少在RNA水平），这可以在治疗上加以利用。然而，抗肌萎缩蛋白杆的生物物理和生物化学结构还没有很好地理解。它传统上被认为是由一些重复的基序和一些散布的所谓的铰链区组成。然而，我已经表明，这些区域相互作用的方式是异质的-一些图案似乎独立于他们的邻居，而其他的则不是，表现出强烈的合作结构相互作用。这对编辑有明显的影响，因为在合作区域中的编辑可以具有远端效应。同样，正常的RNA剪接事件似乎通过外显子跳跃产生天然缺失变体，但模式令人困惑，因为大多数编辑并不发生在这些重复基序的连接处附近（几乎完全用于人为缺失），而是中途，导致新的杂交基序。所有这些因素都导致了产生功能性编辑的抗肌萎缩蛋白变体的不可预测性。我们试图产生一个生物物理和生物化学地图的结构域结构的肌营养不良蛋白杆，从而提供一个合理的基础来编辑这个分子，并更好地了解正常的差异RNA加工产生的推定的变体。 这项工作旨在了解肌营养不良蛋白（Duchenne肌营养不良症中的蛋白质缺陷）的杆区域的结构，这是该蛋白质的最大组成部分，也是导致DMD的大多数突变发生的地方。这个杆在蛋白质和DNA水平上都由许多重复区域组成，它的组装方式表明它可以被编辑以改善这些缺陷。在许多正在开发的治疗策略中，设想了这种杆的编辑和缩短版本;我们将确定如何产生这些缩短的区域，同时仍然保持蛋白质作为一个整体的稳定性。

DESCRIPTION (provided by applicant): The aim of this project is to understand the nature of the rod region of dystrophin. Although this region makes up the bulk of dystrophin's sequence, the much smaller terminal globular domains are often considered more important since the mediate dystrophin's interactions with other cellular components. In many cases, shortened versions of dystrophin in which part of the rod region is removed - the so-called mini- dystrophins - are being studied as possible gene therapy replacements. However, substantial context effects have been seen in animal models, in which slightly differently constructed mini-dystrophins involving different rod modifications have had markedly different efficacies. As well, it has recently been demonstrated that the processing of the dystrophin gene naturally produces various deletions (at least at the RNA level) though exon skipping, which may be therapeutically harnessed. However, the biophysical and biochemical construction of the dystrophin rod is not well understood. It is traditionally thought of as composed of a number of repetitive motifs with a few interspersed so-called hinge regions. However, I have shown that the manner in which these regions interact is heterogeneous - some motifs appear independent of their neighbors, while others are not, exhibiting strongly cooperative structural interactions. This has obvious implications for editing, since editing in a cooperative region can have distal effects. As well, normal RNA splicing events appear to produce natural deletion variants via exon skipping, but the pattern is perplexing, in that most edits do not occur near the junctions of these repeat motifs (as have been nearly exclusively employed in man-made deletions), but midway through, resulting in novel, hybrid motifs. All of these factors contribute to the unpredictability of producing functional edited dystrophin variants. We seek to produce a biophysical and biochemical map of the domain structure of the dystrophin rod, thereby providing a rational basis to edit this molecule, and a better understanding of the putative variants produced by normal differential RNA processing. This work seeks to understand the structure of the rod region of dystrophin (the protein defective in Duchenne Muscular Dystrophy), which is the largest component of this protein, and is where most mutations responsible for DMD occur. This rod is composed of many repetitive regions at both the protein and DNA levels, and the manner in which it is assembled suggests that it can be edited to ameliorate these defects. In many therapeutic strategies under development, edited and shortened versions of this rod are envisioned; and we will determine how to produces these shorted regions while still maintaining stability of the protein as a whole.