Gene therapy for progressive muscular dystrophy using a new generation adenovirus vector and transposase

杜氏肌营养不良症（DMD）是一种致命的进行性肌肉萎缩性疾病，由肌营养不良蛋白基因缺陷引起。除了辅助病毒依赖型腺病毒载体（HDAdv）外，没有病毒载体可以包装14 kb全长的肌营养不良蛋白cDNA，并且HDAdv由于其基因组中的大尺寸缺失而比旧一代腺病毒载体安全得多。我们已经产生了携带myc标记的鼠全长肌营养不良蛋白cDNA（HDAdv-myc-mFLdys）的HDAdv。我们将其注射到7天大的utrophin/dystrophin双敲除小鼠（dko小鼠）的多个近端肌肉中，这些小鼠通常表现出与人类DMD非常相似的症状，因为近端肌肉是DMD患者受影响的器官。注射后8周，转导的肌营养不良蛋白被广泛表达，我们发现中央有核肌纤维显著减少，肌营养不良蛋白相关蛋白、β-肌营养不良蛋白聚糖（p-DG）和α-肌聚糖（a-SG）以及神经元一氧化氮合酶恢复。（nNOS）。被注射的dko小鼠也表现出体重增加，运动能力提高，寿命延长。使用HDAdv，我们可以治疗DMD模型小鼠，即使治疗基因被转移到多个骨骼肌中。因此，多次肌肉注射携带全长dystrophin基因的HDAdv可以减轻DMD患者的症状，弥补功能缺失;由于目的基因产物的表达时间较长，我们尝试利用sleeping beauty转座子系统和HDAdv将dystrophin基因整合到染色体上。然而，它揭示了HDAdv的DNA构建体是线性的，并且它不适合睡美人转座子系统的环状DNA。因此，将dystrophin基因整合到染色体上仍需进一步研究。

Duchenne muscular dystrophy(DMD) is a fatal progressive muscle wasting disease caused by defects in the dystrophin gene. No viral vector except the helper-dependent adenovirus vector(HDAdv) can package 14kb full-length dystrophin cDNA and HDAdv is considerably safer than old-generation adenovirus vectors due to the large-size deletion in its genome. We have generated HDAdv that carries myc-tagged murine full-length dystrophin cDNA(HDAdv-myc-mFLdys). We injected it into the multiple proximal muscles of 7-day-old utrophin/dystrophin double knockout mice (dko mice), which typically show symptoms quite similar to human DMD because the proximal muscles are organs affected in DMD patients. Eight weeks after injections, the transduced dystrophin was widely expressed and we found a significant reduction of centrally nucleated myofibers and the restoration of dystrophin associated proteins, p-dystroglycan (p-DG) and a-sarcoglycan (a-SG), as well as neuronal nitric oxide synthase. (nNOS). The injected dko mice also showed an increase in body weight, an improvement in motor performances, and prolonged lifespan. Using HDAdv, we could treat DMD model mice, even when the therapeutic gene was transferred into multiple skeletal muscles. Our results suggest that multiple intramuscular administrations of HDAdv carrying full-length dystrophin may reduce symptoms and compensate for lost functions in DMD patients.For the long expression of target gene product, we tried to integrate the dystrophin gene into chromosome by using the sleeping beauty-transposone system and HDAdv. However, it revealed that the DNA construct of HDAdv is linear and it does not fit the circular DNA of the sleeping beauty-transposone system. So, further study is necessary to integrate the dystrophin gene into chromosome.