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

我们利用Cre/loxP系统成功地制备并纯化了一种用于基因治疗的新一代腺病毒载体(无胆腺病毒)。这种新一代的腺病毒是辅助子依赖型腺病毒，几乎缺少所有野生腺病毒的DNA，取而代之的是长度超过30kb的非病毒DNA。重复繁殖后，病毒滴度增加。然后用氯化铯浮力密度离心法将扩增载体浓缩到粗裂液的1000倍以上，并对包装DNA的结构和辅助病毒的结构进行了优化，以建立一个稳定的系统，能够产生高效价、低辅助污染的无胆腺病毒。利用该载体可以在体内转导抗肌营养不良蛋白基因在mdx-小鼠胫骨前肌中的表达，由于该载体在基因治疗方面具有很好的应用前景，因此，为了安全的临床应用，必须仔细研究基因的长期表达和免疫的激活。

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.