Recombinant Adeno Associated Virus Vectors for Gene Transfer

DMD是一种X连锁的隐性遗传性疾病，是最常见的遗传性肌病，每3500名男孩中就有一人受到影响。它是由肌细胞结构蛋白Dystrophin基因突变引起的。横纹肌中功能性肌营养不良蛋白的丧失会导致心肌细胞极度脆弱和损伤。骨骼肌不断退化，变得纤维化，脂肪渗出。临床症状首先出现在18个月至3岁的男孩身上。随着疾病的发展，肌肉萎缩会发生，患者在9到12岁之间变得不能行走。死亡的原因是骨骼肌持续丧失，导致脊柱变形、呼吸困难和心肌病。这种疾病总是致命的，通常是在生命的第二个十年，而且总是在生命的第三个十年。目前还没有治愈DMD的方法，目前的治疗方案包括类固醇抗炎药，以限制对肌肉纤维损伤的免疫反应，并仅进行姑息治疗。 重组AAV可用于恢复骨骼肌中肌营养不良蛋白的表达，可以使用蛋白质替代方法，例如表达微肌营养不良蛋白的载体，或者跳过外显子的方法。无论是哪种应用，在临床相关的DMD犬模型中，生产足够数量的rAAV以达到有意义的终点一直是临床试验的主要障碍。我们生产的大多数rAAV载体都包含一个非结构基因，表达一个修饰的U7 RNA，干扰对初级dystrophin转录本的处理。利用特定的反义元件，我们的合作者确定特定的外显子可能从成熟的mRNA中被省略。 这种跳过外显子的方法已经在小鼠DMD模型和犬DMD模型的有限试验中得到了验证。基于这些令人鼓舞的结果，使用表达报告蛋白的rAAV来建立治疗DMD的药理学参数的研究正在进行中。根据这些结果，确定了生物分布、给药途径和媒介毒性。最后，使用报告构建获得的数据，然后可以建立在大型动物DMD模型中产生表型改善的剂量。

DMD, an X-linked recessive disorder, is the most frequent hereditary myopathy affecting approximately 1 in 3500 boys of all races. It is caused by mutations in the gene for the myocyte structural protein dystrophin. The loss of functional dystrophin in striated muscles causes extreme myocyte fragility and damage. The skeletal muscles degenerate continuously becoming fibrotic with adipose infiltrates. Clinical symptoms first manifest in boys between 18 months and 3 years of age. Muscle wasting occurs as the disease progresses and the patients become nonambulatory between 9 to 12 years of age. Mortality results from continuous loss of skeletal muscle causing spinal deformation, breathing difficulties, and cardiomyopathies. The disease is always fatal, often in the second decade and invariably in the third decade of life. There are no cures for DMD, and the current therapeutics regimens involve steroidal anti-inflamatories to limit immune responses to muscle fiber damage and only palliative treatments. Recombinant AAV may be used to restore dystrophin expression to skeletal muscles, either using a protein replacement approach, e.g. vectors that express micro-dystrophin, or an exon-skipping approach. For either application, producing sufficient quantities of rAAV to attain meaningful endpoints in the clinically relevant canine model of DMD has been the major obstacle for clinical trials. Most of the rAAV vector we produce contain a non-structural gene expressing a modified U7 RNA that interferes with processing the primary dystrophin transcript. Using specific anti-sense elements, our collaborators determined that specific exons may be omitted from the mature mRNA. This exon-skipping approach has been validated in murine DMD models and limited trials in canine DMD models. Based on these encouraging results, studies are underway using rAAV that express reporter proteins to establish the pharmacological parameters for treating DMD. From these results, the bio-distribution, routes of administration, vector toxicity are determined. Finally, using the data obtained with reporter constructs, doses that produce phenotypic improvements in large animals DMD models can then be established.