Triple Trans-Splicing Adeno-Associated Virus Vectors Capable of Transferring the Coding Sequence for Full-Length Dystrophin Protein into Dystrophic Mice
Triple Trans-Splicing Adeno-Associated Virus Vectors Capable of Transferring the Coding Sequence for Full-Length Dystrophin Protein into Dystrophic Mice
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DOI：
10.1089/hum.2013.164
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发表时间：
2014-02-01
影响因子：
4.2
通讯作者：
Dickson, George
中科院分区：
文献类型：
--
作者：
Koo, Taeyoung;Popplewell, Linda;Dickson, George
重组腺相关病毒（rAAV）载体已被证明允许非常有效的广泛的转基因表达后，在骨骼肌全身交付，使这些越来越有吸引力的杜氏肌营养不良症（DMD）基因治疗的载体。DMD是一种严重的肌肉消耗性疾病，由DMD基因突变引起，导致肌营养不良蛋白完全丧失。作为DMD的治疗方法，与DMD基因递送相关的主要问题之一是其大的开放阅读框（ORF; 11.1kb）。因此，一系列截短的微小肌营养不良蛋白cDNA（通过单个AAV递送）和微小肌营养不良蛋白cDNA（通过双AAV反式剪接/重叠重建递送）已经在DMD动物模型中进行了广泛测试。然而，关键杆和铰链结构域的肌营养不良蛋白与肌营养不良蛋白相关的蛋白质复合物，如神经元型一氧化氮合酶，syntrophin，和肌营养不良蛋白，相互作用所需的缺失;这些肌营养不良蛋白结构域可能仍然需要纳入增加肌营养不良蛋白的功能和稳定膜刚度。使用AAV载体的全长DMD基因递送仍然难以捉摸，因为单个AAV包装容量有限（4.7kb）。在这里，我们开发了一种新的方法，用于使用三重AAV反式剪接载体系统将全长DMD编码序列递送到营养不良mdx小鼠的骨骼肌中。我们首次报道，三个独立的AAV载体串联携带人DMD编码序列的连续外显子部分，使全长蛋白质的表达成为反式剪接事件的结果，通过其反向末端重复序列共同连接三个载体。这种三重AAV介导的反式剪接方法可适用于将任何大的治疗基因（11 kb ORF）递送到有丝分裂后组织（肌肉或神经元）中，用于治疗各种遗传代谢和遗传疾病。
Recombinant adeno-associated virus (rAAV) vectors have been shown to permit very efficient widespread transgene expression in skeletal muscle after systemic delivery, making these increasingly attractive as vectors for Duchenne muscular dystrophy (DMD) gene therapy. DMD is a severe muscle-wasting disorder caused by DMD gene mutations leading to complete loss of dystrophin protein. One of the major issues associated with delivery of the DMD gene, as a therapeutic approach for DMD, is its large open reading frame (ORF; 11.1kb). A series of truncated microdystrophin cDNAs (delivered via a single AAV) and minidystrophin cDNAs (delivered via dual-AAV trans-spliced/overlapping reconstitution) have thus been extensively tested in DMD animal models. However, critical rod and hinge domains of dystrophin required for interaction with components of the dystrophin-associated protein complex, such as neuronal nitric oxide synthase, syntrophin, and dystrobrevin, are missing; these dystrophin domains may still need to be incorporated to increase dystrophin functionality and stabilize membrane rigidity. Full-length DMD gene delivery using AAV vectors remains elusive because of the limited single-AAV packaging capacity (4.7kb). Here we developed a novel method for the delivery of the full-length DMD coding sequence to skeletal muscles in dystrophic mdx mice using a triple-AAV trans-splicing vector system. We report for the first time that three independent AAV vectors carrying in tandem sequential exonic parts of the human DMD coding sequence enable the expression of the full-length protein as a result of trans-splicing events cojoining three vectors via their inverted terminal repeat sequences. This method of triple-AAV-mediated trans-splicing could be applicable to the delivery of any large therapeutic gene (11kb ORF) into postmitotic tissues (muscles or neurons) for the treatment of various inherited metabolic and genetic diseases.