Gao G, Qu G, Burnham M S, Huang J, Chirmule N, Joshi B, Yu Q C, Marsh J A, Conceicao C M, Wilson J M
Institute for Human Gene Therapy, University of Pennsylvania, Philadelphia, PA 19104, USA.
Hum Gene Ther. 2000 Oct 10;11(15):2079-91. doi: 10.1089/104303400750001390.
Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.
重组腺相关病毒（AAV）在人类基因治疗方面极具前景。尽管有证据表明AAV能在多种不同组织中介导长期基因转移，但大量制备和纯化这种病毒载体的困难仍然是在临床试验中评估AAV载体的主要障碍。目前基于通过氯化铯沉淀的纯化方法不可扩展，且产生的产品质量不足。在本文中，我们报告了一种使用完全封闭的双柱色谱系统纯化AAV的新技术。通过这种方法纯化的AAV载体产量高，效力增强，且柱纯化制剂的纯度有显著提高。我们之前报道了一种基于含AAV Rep/Cap的细胞系（B50）和腺病毒-AAV杂交病毒产生AAV的新方法，该方法适合在生物反应器中放大。通过将我们在此报告的全新可扩展纯化工艺与B50/杂交生产方法相结合，制备出临床和潜在商业应用所需规模和纯度的AAV载体将是可行的。
