Gene therapy for progressive muscular dystrophy using a new generation adenovirus vector (gutless adenovirus)

辅助依赖性腺病毒（HDAd）载体的免疫原性较低，并且具有高达37 kb的较大克隆能力，足以携带全长肌营养不良蛋白cDNA。然而，将抗肌萎缩蛋白转导至成熟肌肉的高和长期表达仍然困难。其主要原因之一是柯萨奇病毒和腺病毒受体（CAR）在成熟肌肉中的表达非常低。我们构建了两个不同的HDAd载体。一个包含LacZ和鼠全长肌营养不良蛋白表达盒（HDAdLacZ-dys），另一个是一种新的，改进的载体，包含CAR和肌营养不良蛋白表达盒（HDAdCAR-dys）。我们最初证明了在新生期期间用HDAdLacZ-dys注射的mdx肌肉中的高肌营养不良蛋白表达和营养不良病理学的预防。此外，我们证明了重复注射HDAdCAR-dys到成熟肌肉中导致肌营养不良蛋白阳性纤维的数量比单次注射多约9倍，从而恢复肌营养不良蛋白相关蛋白的表达。该数据还显示，HDAdCAR-dys使得能够将腺病毒（Ad）载体施用至具有对相同血清型Ad的预先存在的免疫力的宿主。重复注射含有CAR和肌营养不良蛋白表达盒的HDAd载体可以提高后续肌营养不良蛋白基因转移到成熟mdx肌肉的效率。这一结果表明，我们的新HDAd载体将提供一个新的基因治疗策略的杜氏肌营养不良症，提高其他遗传性肌病的基因治疗的前景。

The helper-dependent adenovirus(HDAd) vector is less immunogenic and has a larger cloning capacity of up to 37 kb enough to carry the full-length dystrophin cDNA. However, high and long-term expression of dystrophin transduced to mature muscle still remains difficult. One of the main reasons for this is that the expression of the Coxsakievirus and adenovirus receptor(CAR) is very low in mature muscle. We have constructed two different HDAd vectors. One contains the LacZ and the murine full-length dystrophin expression cassette(HDAdLacZ-dys), and the other is a new, improved vector containing the CAR and the dystrophin expression cassette(HDAdCAR-dys). We initially demonstrated high dystrophin expression and prevention of the dystrophic pathology in mdx muscle injected during the neonatal phase with HDAdLacZ-dys. Furthermore, we demonstrated that repeated injections of HDAdCAR-dys into mature muscle led to approximately nine times greater dystrophin-positive fibers in number than a single injection, thereby recovering the expression of dystrophin-associated proteins. This data has also shown that HDAdCAR-dys enabled administration of adenovirus(Ad) vector to the host with pre-existing immunity to the same serotype of Ad. Repetitive injections of the HDAd vector containing the CAR and the dystrophin expression cassette could improve the efficiency of subsequent dystrophin gene transfer to mature mdx muscle. This result suggests that our new HDAd vector will provide a novel gene therapy strategy for Duchenne muscular dystrophy, raising the prospects for gene therapy of other hereditary myopaties.