Recombinant Adeno Associated Virus Vectors for Gene Transfer

DMD是一种x连锁隐性遗传病，是最常见的遗传性肌病，在所有种族中，大约每3500名男孩中就有1人患此病。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会导致心肌细胞极度脆弱和损伤。骨骼肌不断退化，纤维化并伴有脂肪浸润。临床症状首先表现在18个月至3岁的男孩身上。肌肉萎缩随着疾病的进展而发生，患者在9至12岁之间变得不能走动。死亡是由于骨骼肌的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，通常发生在第二个十年，而总是发生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，以限制对肌纤维损伤的免疫反应，而且只有姑息性治疗。

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.