Gene Therapy for Muscular Dystrophy

杜氏肌营养不良症（DMD）是一种由dystrophin基因突变引起的X连锁隐性遗传性肌营养不良症。它引起骨骼肌和心肌的进行性肌病，导致患者在早期残疾和过早死亡。目前尚无有效的治疗DMD的方法。基因治疗在DMD的治疗中已显示出其可行性和良好的应用前景。然而，在成功地将基因治疗应用于DMD患者之前，仍存在许多障碍。由于大多数载体对转基因的包装能力有限，肌营养不良蛋白基因的大尺寸（14 kb的cDNA）是成功的DMD基因治疗的主要障碍之一。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）来修复大尺寸FVIII基因和相应的血友病A表型校正。由于SMaRT仅替换基因的突变部分，因此无需递送基因的整个cDNA，从而避免了在基因治疗载体中递送大基因（例如，FVIII和肌营养不良蛋白）的潜在低效率。此外，SMaRT纠正内源性突变的肌营养不良蛋白基因，从而在基因治疗后诱导针对肌营养不良蛋白的最小潜在免疫。在本申请中，我们提出测试用于DMD基因治疗的基于SMaRT的RNA修复。我们的长期目标是开发一种成功的DMD基因治疗方法。本提案的目的是测试基于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修复来纠正突变型dystrophin基因并恢复dystropin活性，这将为DMD的基因治疗开辟一条新途径，这也可能适用于由大尺寸基因缺陷引起的其他遗传疾病。我们在本申请中提出两个具体目标：1.设计和构建肌营养不良蛋白特异性的前反式剪接分子（PTM），以促进肌营养不良蛋白突变的修复; 2.为了测试基于SMaRT的RNA修复在通过AAV载体递送肌营养不良蛋白特异性PTM后恢复营养不良转基因小鼠模型（mdx 4cv小鼠）中肌营养不良蛋白活性的可行性和效率。PHS 398/2590（Rev. 09/04）Page Continuation Format Page主要研究者/项目负责人（末，首，中）：Chao，Hengjun杜氏肌营养不良症（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基因治疗方法的可行性和有效性。PHS 398/2590（2004年9月修订，2006年4月重新发布）

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)