Recombinant Adeno Associated Virus Vectors for Gene Transfer

DMD是一种X连锁隐性遗传疾病，是最常见的遗传性肌病，影响所有种族中约3500名男孩中的1名。它是由肌细胞结构蛋白肌营养不良蛋白基因突变引起的。横纹肌中功能性肌营养不良蛋白的缺失导致肌细胞极度脆弱和损伤。骨骼肌不断退化，纤维化，脂肪浸润。临床症状首先出现在18个月至3岁的男孩中。肌肉萎缩随着疾病的进展而发生，患者在9至12岁之间变得不能行走。骨骼肌的持续丧失导致脊柱变形、呼吸困难和心肌病，从而导致死亡。这种疾病总是致命的，往往在生命的第二个十年，总是在第三个十年。DMD没有治愈的方法，目前的治疗方案包括类固醇抗炎药，以限制对肌纤维损伤的免疫反应，并且只有姑息治疗。 重组AAV可用于恢复肌营养不良蛋白在骨骼肌中的表达，或者使用蛋白质替代方法，例如表达微小肌营养不良蛋白的载体，或者使用外显子跳跃方法。对于任一应用，产生足够量的rAAV以在DMD的临床相关犬模型中获得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载体含有表达修饰的U7 RNA的非结构基因，该修饰的U7 RNA干扰初级肌营养不良蛋白转录物的加工。使用特定的反义元件，我们的合作者确定特定的外显子可以从成熟的mRNA中省略。 这种外显子跳跃方法已在鼠DMD模型中得到验证，并在犬DMD模型中进行了有限的试验。基于这些令人鼓舞的结果，正在进行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。根据这些结果，确定了生物分布、给药途径、载体毒性。最后，使用用报道构建体获得的数据，然后可以建立在大动物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.