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

辅助腺病毒（HDAd）载体具有较低的免疫原性，具有较大的克隆能力，可达37 kb，足以携带全长抗肌营养不良蛋白cDNA。然而，长期高表达的肌营养不良蛋白转导到成熟肌肉仍然是困难的。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。我们已经构造了两个不同的HDAd向量。一种是含有LacZ和小鼠全长肌营养不良蛋白表达盒（HDAdLacZ-dys），另一种是含有CAR和肌营养不良蛋白表达盒（HDAdCAR-dys）的新型改良载体。我们最初证明了在新生儿期注射HDAdLacZ-dys的mdx肌肉中，抗营养不良蛋白的高表达和预防营养不良病理。此外，我们证明，在成熟肌肉中反复注射hddad -dys，导致肌营养不良蛋白阳性纤维的数量比单次注射增加了约9倍，从而恢复了肌营养不良蛋白相关蛋白的表达。这些数据还表明，hdcar -dys能够将腺病毒（Ad）载体施用于对相同血清型Ad具有预先免疫的宿主。重复注射含有CAR和肌营养不良蛋白表达盒的HDAd载体可以提高随后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载体将为杜氏肌营养不良症提供一种新的基因治疗策略，为其他遗传性肌病的基因治疗提供前景。

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.