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

辅助依赖型腺病毒(HDAd)载体的免疫原性较弱，克隆能力较大，可达37kb，足以携带全长dystrophin基因。然而，转导到成熟肌肉的dystrophin的高和长期表达仍然是困难的。其中一个主要原因是柯萨基病毒和腺病毒受体(CAR)在成熟肌肉中的表达很低。我们构建了两个不同的HDAd载体。一个包含LacZ和小鼠全长dystrophin表达盒(HDAdLacZ-dys)，另一个是包含CAR和dystrophin表达盒的新的改进载体(HDAdCAR-dys)。我们最初证明，在新生儿期注射HDAdLacZ-dys的MDX肌肉中，dystrophin的高表达和营养不良病理的预防。此外，我们还证明，在成熟肌肉中重复注射HDAdCAR-dys后，dystrophin阳性纤维的数量大约是单次注射的九倍，从而恢复了dystrophin相关蛋白的表达。这一数据还表明，HDAdCAR-dys能够将腺病毒(Ad)载体接种到对同一血清型Ad预先存在免疫力的宿主。重复注射含有CAR和dystrophin表达框的HDAd载体可以提高随后将dystrophin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Duchenne肌营养不良症提供一种新的基因治疗策略，为其他遗传性近视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.