Gene Therapy for Muscular Dystrophy

杜氏肌营养不良症（DMD）是一种由肌营养不良蛋白基因突变引起的x连锁隐性肌肉疾病。它可引起骨骼肌和心肌的进行性肌病，导致患者早期残疾和过早死亡。迄今为止，对DMD还没有有效的治疗方法。基因治疗在DMD的治疗中已显示出其可行性和良好的潜力。然而，在成功地将基因治疗应用于DMD患者之前，仍然存在许多障碍。由于大多数载体对转基因的包装能力有限，抗肌萎缩蛋白基因（14 kb cDNA）的大尺寸是成功治疗DMD基因的主要障碍之一。我们最近报道了通过使用一种新的RNA修复策略：剪接体介导的RNA反式剪接（SMaRT）修复大尺寸FVIII基因和随之而来的血友病A表型纠正。由于SMaRT只替换基因的突变部分，它避免了传递基因的整个cDNA的需要，因此避免了在基因治疗载体中传递大基因（如FVIII和肌营养不良蛋白）的潜在低效率。此外，SMaRT纠正了内源性突变的肌营养不良蛋白基因，因此它应该在基因治疗后诱导对肌营养不良蛋白的最小潜在免疫。在这个应用中，我们建议测试基于smart的RNA修复用于DMD基因治疗。我们的长期目标是开发一种成功的DMD基因治疗方法。本研究的目的是测试基于smart的RNA修复在DMD基因治疗中的可行性和效率。该项目的基本原理是，通过基于smart的RNA修复来纠正突变的肌营养不良蛋白基因并恢复肌营养不良蛋白的活性，将为DMD的基因治疗开辟一条新的途径，这也可能适用于其他由大尺寸基因缺陷引起的遗传疾病。我们在这个应用中提出了两个具体的目标：1。设计和构建抗肌营养不良蛋白特异性预反式剪接分子（PTM），促进抗肌营养不良蛋白突变的修复；2. 在营养不良转基因小鼠模型（mdx4cv小鼠）中，通过AAV载体传递营养不良蛋白特异性PTMs后，测试基于smart的RNA修复恢复营养不良蛋白活性的可行性和效率。PHS 398/2590 （Rev. 09/04）首席研究员/项目主任（最后，第一，中）：Chao, Hengjun Duchenne muscular dystrophy （DMD）是最常见的遗传性疾病之一。DMD没有有效的治疗方法。本申请的目的是测试一种新的DMD基因治疗程序的可行性和效率。PHS 398/2590（09/04修订，4/2006重新发布）

Duchenne muscular dystrophy (DMD) is an X-linked recessive muscular disease resulting from mutation of dystrophin gene. It causes progressive myopathy of skeletal and cardiac muscle, leading to disability at early age and premature death of the patients. There is no effective treatment for DMD to date. Gene therapy has demonstrated its feasibility and promising potential in treatment for DMD. However, numerous obstacles remain before successful application of gene therapy to DMD patients. As most vectors have a limited packaging capacity for transgenes, the large size of the dystrophin gene (14 kb of cDNA) is one of the major hurdles in successful DMD gene therapy. We recently reported repair of the large size FVIII gene and the consequential hemophilia A phenotype correction by using a novel RNA repair strategy: splicesome- mediated RNA trans-splicing (SMaRT). Because SMaRT replaces only the mutated part of a gene, it obviates the need to deliver the entire cDNA of the gene, and therefore circumvents the potential inefficiency of delivering the large genes such as FVIII and dystrophin in a gene therapy vector. In addition, SMaRT corrects the endogenous mutated dystrophin gene, it thereby should induce minimal potential immunity against dystrophin following gene therapy. In this application, we propose to test SMaRT-based RNA repair for DMD gene therapy. Our long-term goal is to develop a successful gene therapy procedure for DMD. The objective of this proposal is to test feasibility and efficiency of SMaRT-based RNA repair in DMD gene therapy. The rationale underlying this project is that correction of mutant dystrophin gene and restoration of dystropin activity by SMaRT-based RNA repair will open a novel avenue to gene therapy for DMD, which also can be potentially applicable to other genetic diseases caused by defects in genes of large size. We propose two specific aims in this application: 1. To design and build dystrophin-specific Pre-Trans-splicing Molecules (PTM) that facilitate repair of the dystrophin mutation; 2. To test feasibility and efficiency of SMaRT-based RNA repair in restoring dystrophin activity in a dystrophy transgenic mouse model (mdx4cv mice) following delivery of the dystrophin- specific PTMs by AAV vectors. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page Principal Investigator/Program Director (Last, First, Middle): Chao, Hengjun Duchenne muscular dystrophy (DMD) is one of the most frequent genetic diseases. There is no effective treatment for DMD. The objective of this application is to test feasibility and efficiency of a novel gene therapy procedure for DMD. PHS 398/2590 (Rev. 09/04, Reissued 4/2006)