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

 描述（由申请人提供） 基因疗法是治疗杜氏肌营养不良症 (DMD) 的一种有前途的方法。然而，目前的方法通常需要在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来遗传物质，以暂时恢复肌营养不良蛋白的表达，这两者都具有显着的安全性和实际问题。此外，这些策略因无法递送大而复杂的肌营养不良蛋白基因序列而受到限制。这些基因替代方法的一个有吸引力的替代方法是内源性突变肌营养不良蛋白基因的靶向修复。这一概念被称为基因组编辑，代表了一种治疗 DMD 的潜在方法，而无需永久整合或反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，从源头上纠正了问题。最近革命性技术的发展使基因组编辑已成为人类基因治疗的现实，这些技术使用工程酶在基因组的特定位点剪切和粘贴 DNA 序列。事实上，基因组编辑目前正处于治疗癌症和艾滋病毒的临床试验中。最近开发的基因组编辑技术（称为 CRISPR）比以前的技术更加强大，并且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。目前正在进行多项利用 CRISPR 纠正遗传性疾病的努力，我们已经证明可以恢复 DMD 患者肌肉细胞中肌营养不良蛋白的表达。然而，为了使其能够用于临床转化，我们必须 在该疾病的动物模型中展示了骨骼和心肌组织中成功的基因组编辑。在这项研究中，我们将使用腺相关病毒将 CRISPR 递送至 DMD 小鼠模型和携带人类肌营养不良蛋白基因的小鼠模型的骨骼和心肌。该研究计划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 小鼠模型中肌营养不良蛋白的正确表达和功能。该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发挥的潜力，解决了传统基因疗法的根本局限性，以及安全有效永久治愈 DMD 的未满足需求。重要的是，这种方法还广泛适用于除 DMD 之外的多种遗传疾病。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完善，以广泛造福于受遗传性疾病影响的患者。最后，骨骼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学研究。

 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.