CRISPR/Cas9-Based Gene Editing for the Correction of Duchenne Muscular Dystrophy

 描述（由申请人提供） 基因治疗是治疗杜氏肌营养不良症（DMD）的一种很有前途的方法。然而，目前的方法通常需要向基因组中添加额外的肌营养不良蛋白基因或终身重新施用外源遗传物质，其瞬时起作用以恢复肌营养不良蛋白表达，这两者都具有显著的安全性和实际问题。此外，这些策略受到无法递送大而复杂的肌营养不良蛋白基因序列的限制。这些基因替代方法的一个有吸引力的替代方案是内源性突变型肌营养不良蛋白基因的靶向修复。这种被称为基因组编辑的概念代表了DMD的潜在治愈方法，而无需永久整合或重复暴露于外来生物材料。此外，它通过纠正天然存在的肌营养不良蛋白基因的突变来纠正源头问题。基因组编辑已经成为人类基因治疗的现实，这是通过最近开发的变革性技术实现的，这些技术使用工程酶在基因组中的特定位点切割和粘贴DNA序列。事实上，基因组编辑现在正在进行治疗癌症和艾滋病毒的临床试验。最新开发的基因组编辑技术CRISPR比以前的技术更加强大，在不到两年的时间里迅速改变了生物医学研究和生物技术的所有领域。目前正在努力使用CRISPR来纠正遗传疾病，我们已经证明有可能恢复DMD患者肌肉细胞中的肌营养不良蛋白表达。然而，为了使其适用于临床翻译，我们必须 在该疾病的动物模型中，在骨骼肌和心肌组织中展示成功的基因组编辑。在这项研究中，我们将使用腺相关病毒将CRISPR递送到DMD小鼠模型和携带人肌营养不良蛋白基因的小鼠模型的骨骼肌和心肌。这项研究提案的总体目标是开发通过体内靶向基因组编辑恢复肌营养不良蛋白表达的方法。核心假设是核酸酶介导的基因校正将导致DMD小鼠模型中适当的肌营养不良蛋白表达和功能。这项研究计划具有创新性，因为它利用了CRISPR基因组编辑技术尚未实现的潜力，以解决传统基因疗法的根本局限性以及对DMD安全有效的永久治疗的未满足需求。重要的是，这种方法也广泛适用于除了DMD之外的许多遗传疾病。因此，除了确定用于治疗DMD的主要候选核酸酶和递送方法外，这项工作还将导致CRISPR技术的进一步开发和改进，以广泛造福受遗传性疾病影响的患者。最后，骨骼肌和心肌体内基因组编辑技术的发展将广泛用于生物技术和基础科研。

 DESCRIPTION (provided by applicant) Gene therapy is a promising approach to treating Duchenne Muscular Dystrophy (DMD). However, current methods typically require the addition of extra dystrophin genes to the genome or the lifelong re- administration of foreign genetic material that works transiently to restore dystrophin expression, both of which have significant safety and practical concerns. Furthermore, these strategies have been limited by an inability to deliver the large and complex dystrophin gene sequence. An appealing alternative to these gene replacement approaches is the targeted repair of the endogenous mutant dystrophin gene. This concept, known as genome editing, represents a potential cure to DMD without the need for permanent integration of or repeated exposure to foreign biological material. Furthermore, it corrects the problem at the source by correcting the mutation to the naturally occurring dystrophin gene. Genome editing has been made a reality for human gene therapy by the recent development of transformative technologies that use engineered enzymes to cut and paste DNA sequences at specific sites in the genome. In fact, genome editing is now in clinical trials for treating cancer and HIV. The most recently developed genome editing technology, known as CRISPR, is much more robust than previous technologies and has rapidly transformed all areas of biomedical research and biotechnology in less than two years. Several efforts are underway to use CRISPR to correct genetic diseases, and we have demonstrated that it is possible to restore dystrophin expression in muscle cells from DMD patients. However, for this to be viable for clinical translation, we must demonstrate successful genome editing in skeletal and cardiac muscle tissue in animal models of the disease. In this study, we will use adeno- associated virus to delivery CRISPR to skeletal and cardiac muscles of a mouse model of DMD and a mouse model carrying the human dystrophin gene. The overall objective of this research proposal is to develop methods to restore dystrophin expression via targeted genome editing in vivo. The central hypothesis is that nuclease-mediated gene correction will lead proper dystrophin expression and function in mouse models of DMD. This research plan is innovative because it capitalizes on the unfulfilled potential of the CRISPR genome editing technology to address the fundamental limitations of conventional gene therapies and the unmet need for a safe and effective permanent cure to DMD. Importantly, this approach is also broadly applicable to numerous genetic diseases in addition to DMD. Thus in addition to identifying a lead candidate nuclease and delivery method for treatment of DMD, this work will also lead to additional development and refinements of the CRISPR technology to broadly benefit patients affected by hereditary disorders. Finally, the development of technologies for in vivo genome editing in skeletal and cardiac muscle will be broadly useful for biotechnology and basic scientific research.