Project 1: Translational and pre-clinical studies of muscular dystrophy gene therapy using AAV

本翻译研究项目旨在改进目前AAV载体介导的抗肌营养不良蛋白基因 治疗方法，使这些方法适用于显性肌营养不良症，并提高我们的临床 基础设施，以促进参与未来的AAV基因治疗试验。我们之前的努力涉及到 多年来的合作研究，在基因治疗方面具有显著先进的方法和途径 DMD。它们还导致了开始利用抑制性RNAi和基因靶向来消除 显性遗传性肌营养不良的基因表达。我们还开发了一些方法来 利用AAV载体进行系统基因传递，并在肌肉基因研究方面取得了重要进展 在AAV载体传递后，允许肌肉限制的基因表达和免疫逃避的调节。 AAV/微肌营养不良蛋白的方法已经足够远，它们正在进入人体临床试验 评估安全性，主要是在骨骼肌中的疗效。然而，微克隆仍然没有完全 与骨骼肌相比，心肌的功能和活性降低。基因治疗 由于缺乏良好的动物模型，以及难以使RNAi适应 AAV。尽管如此，这些方法还远远不够，因此有理由和迫切需要加强其 应用于多种不同类型肌营养不良症的最佳基因治疗。因此，我们的具体情况 目的集中在当前DMD和FSHD基因治疗方法的几个关键限制。为 作为DMD，我们将重点放在增强功能上针对骨骼肌和心肌的方法上。我们 将开发在所有横纹肌中有效的改进的基因调节盒，以及仅在骨骼中有效 或者是心肌。在横纹肌中具有增强功能的新型微型和微肌营养不良蛋白将被 在多个测试系统中进行设计和筛选。我们还将测试一种有前景的双矢量策略，结合 肌营养不良蛋白置换(基于结构的治疗)，通过增加 核糖核苷酸还原酶(收缩增强疗法)以改善心脏功能。我们的第二个领域 重点是建立在之前针对DMD的AAV研究和FSHD适应的小鼠模型开发的基础上 AAV方法用于FSHD的基因治疗。这些研究将包括对Dux4表达模式的分析 肌肉细胞。他们还将专注于开发和测试AAV载体介导的组织特异性表达 靶向Dux4基因的RNAi发夹在AAV-DUX4小鼠中低水平表达的研究 模特。最后，我们计划开发专注于心脏功能的患者数据库和自然病史数据 西雅图丙二醛诊所的同事们。我们将从正在进行的心脏成像中收集和组织数据 接受服务的DMD患者群体的研究、功能读数、自然病史数据和突变谱 西雅图诊所。这些研究将扩大项目2的试验准备目标，将DMD患者包括在内。

This translational research project is designed to improve current AAV vector mediated dystrophin gene therapy methods, to adapt those methods for dominant muscular dystrophies, and to enhance our clinical infrastructure to facilitate participation in future AAV gene therapy trials. Our previous efforts have involved years of collaborative studies that have significantly advanced methods and approaches for gene therapy of DMD. They have also led to approaches to begin harnessing inhibitory RNAi and gene targeting to abrogate gene expression in dominantly inherited muscular dystrophies. We have also developed approaches for systemic gene delivery using AAV vectors, and have made important advances in the study of muscle gene regulation that enable muscle-restricted gene expression and immune evasion following AAV vector delivery. The AAV/micro-dystrophin approaches are far enough along that they are entering human clinical trials to assess safety, and primarily in skeletal muscles, efficacy. The micro-clones, however, are still not fully functional and show reduced activity in cardiac muscles compared with skeletal muscles. The gene therapy approaches for FSHD have been slowed by a lack of good animal models and difficulties in adapting RNAi to AAV. Nonetheless these methods are far enough along that it is reasonable and imperative to enhance their application for optimal gene therapy of many different types of muscular dystrophy. Consequently, our specific aims focus on several of the key limitations of current approaches to gene therapy for DMD and FSHD. For DMD, we focus on enhancing approaches that will functionally target both skeletal and cardiac muscles. We will develop improved gene regulatory cassettes active in all striated muscles, as well as exclusively in skeletal or cardiac muscle. Novel mini-and micro-dystrophins with enhanced function in striated muscles will be designed and screened in multiple test systems. We will also test a promising dual vector strategy, combining dystrophin replacement (structural-based therapy) with enhanced contractile performance via increased ribonucleotide reductase (contractile augmentation therapy) to improve cardiac performance. Our second area of focus is to build upon previous AAV studies for DMD and on mouse model development for FSHD to adapt AAV methods for gene therapy of FSHD. These studies will include analysis of patterns of Dux4 expression in muscle cells. They will also focus on developing and testing AAV vector mediated tissue-specific expression of RNAi hairpins targeting Dux4 mRNA in the context of low level mRNA expression in our AAV-DUX4 mouse model. Finally, we plan to develop patient databases and natural history data focused on cardiac function with colleagues at the MDA clinics in Seattle. We will gather and organize data from ongoing cardiac imaging studies, functional readouts, natural history data and mutational spectra in the DMD patient populations served by the Seattle clinics. These studies will expand the trial readiness goals of Project 2 to include DMD patients.