GENE THERAPY FOR DUCHENNE MUSCULAR DYSTROPHY

杜氏肌营养不良症是一种进行性和致命的X连锁肌病 由位于Xp 21的Dystrophin基因的突变和缺失引起。 DMD的治疗由于需要诱导抗肌萎缩蛋白而变得复杂 在广泛的骨骼肌和心脏中表达。到目前为止， 这是不可能使用基于细胞的疗法或化学疗法 转染方法。因此，最近的兴趣集中在使用 以病毒为基础的体细胞基因治疗方法， DMD患者骨骼肌和心肌中肌营养不良蛋白的表达。 在本申请中描述的研究中，我们建议使用系统性的 复制缺陷型腺病毒载体的递送以编程骨骼 肌肉和心脏特异性表达的肌营养不良蛋白小基因， DMD大动物模型。腺病毒载体被选择用于这些 研究，因为包括我们自己在内的几个小组最近的工作 表明它们代表了目前唯一可用的病毒 能够有效编程重组体的递送系统 基因表达在很大比例的非复制骨骼和 全身给药后体内心肌细胞。我们 拟议的研究将利用骨骼肌和心脏特异性 转录调控元件，其先前克隆并表征为 我们的实验室将肌营养不良蛋白基因的表达限制在 合适的肌肉细胞类型。为了规避技术 与使用14 kb肌营养不良蛋白cDNA相关的困难，我们将使用 一种较小的、天然存在的肌营养不良蛋白cDNA缺失突变体。的 通过复制的系统性施用治疗DMD的可行性- 有缺陷的腺病毒最初将在xmd狗中进行评估， 我们已经证明了一个肌肉萎缩症的模型， 遗传、组织病理学和功能特征， 那些人类疾病。在本提案所述的研究中，我们 计划（i）产生一系列重组的、复制缺陷的 含有萤火虫荧光素酶、细菌lacZ或 在心脏和骨骼肌调控下的人小肌萎缩蛋白基因 上述肌肉特异性调节元件，（ii）优化 在新生狗中施用这些载体的方法，（iii） 将表达肌养蛋白的腺病毒载体给予xmd狗，和（iv） 评估这种病毒对疾病进展和骨骼 和心肌功能。此外，我们会审慎评估 腺病毒给药的安全性和潜在副作用 动物这些研究将对DMD的治疗有直接的意义。 此外，它们将对以下疾病的治疗产生重要影响： 其他各种遗传性肌病

Duchenne Muscular Dystrophy is a progressive and lethal X-linked myopathy caused by mutations and deletions in the Dystrophin gene located at Xp21. The treatment of DMD has been complicated by the need to induce dystrophin expression in a wide range of skeletal muscles and in the heart. Thus far, this has been impossible using cell-based therapies or chemical transfection approaches. Therefore, recent interest has focused on the use of viral-based somatic gene therapy approaches to program recombinant dystrophin expression in the skeletal and cardiac muscle of DMD patients. In the studies described in this application, we propose to use systemic delivery of replication-defective adenovirus vectors to program skeletal muscle- and cardiac-specific expression of a dystrophin mini gene in a large animal model of DMD. Adenovirus vectors were chosen for these studies because recent work from several groups including our own has demonstrated that they represent the only currently available viral delivery system that is capable of efficiently programming recombinant gene expression in a large percentage of non-replicating skeletal and cardiac muscle cells in vivo following systemic administration. Our proposed studies will make use of skeletal muscle- and cardiac-specific transcriptional regulatory elements previously cloned and characterized by our laboratory to restrict expression of the dystrophin gene to the appropriate muscle cell types. In order to circumvent technical difficulties associated with use of the 14 kb dystrophin cDNA, we will use a smaller, naturally-occurring deletion mutant of the dystrophin cDNA. The feasibility of treating DMD by the systemic administration of replication- defective adenoviruses will initially be assessed in xmd dogs, a canine model of muscular dystrophy that has been demonstrated by us to display genetic, histopathological and functional features that closely resemble those of the human disease. In the studies described in this proposal, we plan to (i) generate a series of recombinant, replication-defective adenoviruses containing either the firefly luciferase, bacterial lacZ, or human minidystrophin genes under the control of the cardiac and skeletal muscle-specific regulatory elements described above, (ii) optimize the method of administration of these vectors in neonatal dogs, (iii) administer dystrophin-expressing adenovirus vectors to xmd dogs, and (iv) assess the effects of this virus on disease progression and on skeletal and cardiac muscle function. In addition, we will carefully assess the safety and potential side effects of adenovirus administration in these animals. These studies will have direct relevance to the therapy of DMD. Additionally, they will have important implications for the treatment of a variety of other inherited myopathies.