Study on the production and purification of a new generation adenovirus vector (gutless adenovirus) and its clinical application

利用Cre/loxP系统成功制备并纯化了新一代用于基因治疗的腺病毒载体（无肠腺病毒）。这种新一代腺病毒是辅助依赖型腺病毒，几乎缺失所有的野生型腺病毒DNA，而是被替换为长度超过30 kb的非病毒DNA。经反复传代后病毒滴度增加。然后用CsCl浮力密度离心法将增殖的载体浓缩到粗裂解物的1000倍以上。我们优化了包装DNA的结构和辅助病毒的结构，以建立一个能够生产高滴度无肠腺病毒原液且辅助病毒污染低的稳定系统。此外，我们构建了一种无肠腺病毒，其含有16.5kb长的小鼠全长肌营养不良蛋白和2kb长的人柯萨奇病毒和腺病毒受体（CAR）的表达单位。利用该载体可以在体内转导mdx小鼠胫骨前肌中dystrophin的表达，该载体在基因治疗中具有很好的应用前景，但为了安全地应用于临床，必须对长期基因表达和免疫激活进行仔细研究。

We successfully produced and purified a new generation adenovirus vector (gutless adenovirus) aiming for gene therapy by using Cre/loxP system. This new generation adenovirus is helper-dependent adenovirus and lack virtually almost all of the wild adenoviral DNA.Instead of the deletion it was replaced to non-viral DNA longer than 30kb. After the repetitive propagations viral titer increased. Then propagated vectors could be condensed by CsCl buoyant density centrifugation to 1000-fold higher than that of crude lysate.And we optimized the structure of packaging DNA and the structure of helper virus for a robust system capable of producing high-titer gutless adenovirus stocks with low helper contamination.Furthermore, we constructed a gutless adenovirus containing the expression units of 16.5kb-long mouse full-length dystrophin and 2kb-long human Coxsackievirus and adenovirus receptor (CAR). By using this vector we could transduced the expression of dystrophin in tibialis anterior skeletal muscles of mdx-mouse in vivo.As this vector is very promising for gene therapy, long-term gene expression and activation of immunity must be carefully studied for safe clinical application.