A Novel Technology for Full-Length Gene Replacement Therapy of Duchenne Muscular Dystrophy

项目摘要和摘要 Duchenne肌营养不良症(DMD)是一种遗传性疾病，由基因的无义突变引起 Dstrophin基因，DMD。肌营养不良蛋白在保护肌肉免受机械性损伤和 该基因的突变会导致肌肉退化。这种疾病的自然发展涉及肌肉 四肢退变导致心脏和横隔肌丧失活动能力并最终退化， 导致死亡。杜氏肌营养不良症影响5000名男婴中的1名，导致大多数患者瘫痪 到12岁时，平均会导致26岁前死亡。目前还没有已知的治愈方法，目前的治疗只是延缓了 疾病进展或旨在改善生活质量。 基因治疗是一项旨在治愈遗传病的新兴技术。目前，有两种主要方法 正在探索中。尽管CRISPR/CAS9的发展大大推进了基因工程在 近年来，对DMD等疾病的临床治疗仍然存在许多障碍，包括非靶点编辑、 插入/缺失突变、大蛋白复合体的传递以及表达的免疫原性 细菌酶。基因替代疗法是一种很有前景的替代策略，也是一种潜在的治愈策略。基因- 腺相关病毒(AAV)提供的替代疗法提供了一种优雅的解决方案，它通过取代 而不是编辑有缺陷的基因。利用AAVs，外源DNA被输送到靶细胞并稳定地驻留 染色体外的。随后可以表达抗肌营养不良蛋白基因，在功能上取代 有缺陷的内源性拷贝。然而，由于AAVs的货运能力限制，目前的临床测试 仅限于Dystrophin的高度截断版本，导致疗效有限。 最近开发的技术克服了自动驾驶飞机载货能力的限制，为 全长基因替换的交付。首先，分离基因的构建是通过将全基因分割成 片段，两侧是包含内含子和碱基配对结合序列的区域，其中每个片段 是由AAV鸡尾酒的一个亚群提供的。在细胞内，表达的RNA片段是局部的 通过结合结构域的碱基配对稳定，然后进行剪接体介导的连接反应， 从而将这些片段连接成全长的mRNA，用于翻译和恢复功能蛋白。 初步数据表明，这种方法能够提供三片段荧光记者 老鼠的肌肉。这项拟议的研究旨在开发这种技术，作为一种基因替代疗法，通过 构建强健的肌营养不良蛋白基因传递工程，优化靶上和靶外传递 在体外和体内，以及在杜兴的治疗性和非预期宿主反应的特征 肌营养不良症小鼠模型。这个项目将展示一类新的治疗潜力 基因替代疗法。此外，该项目为未来对高等哺乳动物的研究奠定了基础， 用于未来治疗和治愈Duchenne肌营养不良症患者的潜在方法。

Project Summary and Abstract Duchenne muscular dystrophy (DMD) is a genetic disease which arises from a nonsense mutation of the dystrophin gene, dmd. Dystrophin has important functions in protecting muscle from mechanical damage and mutations in the gene results in muscle degeneration. The natural progression of the disease involves muscle degeneration in the limbs leading to loss of mobility and eventual degeneration of cardiac and diaphragm muscle, leading to death. Duchenne muscular dystrophy affects 1 in 5,000 male births, renders most patients paralyzed by age 12, and on average results in death by age 26. There is no known cure and current treatments only delay disease progression or are aimed at quality-of-life improvements. Gene therapy is an emerging technology that aims to cure genetic diseases. Currently, two main approaches are being explored. Although CRISPR/Cas9 developments have advanced genetic engineering significantly in recent years, many barriers still remain to clinical treatment of diseases such as DMD, including off-target editing, insertion/deletion mutations, delivery of the large protein complex, and immunogenicity of expressing the bacterial enzyme. Gene-replacement therapy is a promising alternate and potentially curative strategy. Gene- replacement therapy delivered by Adeno-Associated Virus (AAV) offers an elegant solution by replacing, rather than editing, the defective gene. Using AAVs, exogenous DNA is delivered to target cells and stably resides extra-chromosomally. The dystrophin gene can then be subsequently expressed, functionally replacing the defective endogenous copy. Due to cargo-capacity limitations of AAVs however, current clinical testing have been restricted to highly truncated versions of dystrophin with resulting limited efficacy. Recently developed technology overcomes the cargo capacity limitation of AAVs, providing an avenue for delivery of full-length gene replacements. First, split-gene constructs are designed by dividing the full gene into fragments, flanked by regions containing both intronic and base-pairing binding sequences, where each fragment is delivered by a sub-population of an AAV cocktail. Within the cell, the expressed RNA fragments are locally stabilized by the base-pairing of the binding domains, then undergo a spliceosome-mediated joining reaction, thus concatenating the fragments into full-length mRNA for translation and restoration of functional protein. Preliminary data demonstrates the ability of this approach to deliver three-fragment fluorescent reporters in mouse muscle. The proposed research aims to develop this technology as a gene-replacement therapy through the engineering of robust constructs for dystrophin gene delivery, optimization of on- and off-target delivery both in vitro and in vivo, and characterization of both therapeutic and unintended host responses in a Duchenne Muscular Dystrophy mouse model. This project will demonstrate the therapeutic potential of a novel class of gene-replacement therapies. Further, this project lays the groundwork for future studies in higher mammals, and for a potential future treatment and cure of Duchenne Muscular Dystrophy in patients.