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

新一代腺病毒载体（无肠腺病毒）的制备、纯化及其临床应用研究

• 批准号: 11670631
• 负责人: UCHINO Makoto
• 金额: $ 2.3万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670631/
• 关键词: Gene therapy; gutless adenovirus; Cre; loxP system; recombinant adenovirus vector; skeletal muscle; repetitive transduction of therapeutic gene; coxsackie B and adenovirus receptor; 遺伝子治療; loxPシステム; 新世代のアデノウイルスベクター; 免疫原性; hCAR; 反復遺伝子導入; 遺伝子導

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.

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。