Hildinger Markus, Baldi Lucia, Stettler Matthieu, Wurm Florian M
ExcellGene S.A, Route de l'ile-au-bois 1A, Monthey, Valais, 1870, Switzerland.
Biotechnol Lett. 2007 Nov;29(11):1713-21. doi: 10.1007/s10529-007-9441-3. Epub 2007 Jul 17.
Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required. Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK 293 cells in the presence of serum. In this study, we present a helper- and serum-free production method of AAV vectors in suspension-adapted HEK 293 cells. The method is based on plasmid transfection with 25 kDa linear polyethyleneimine. Compared to existing methods, our system is highly scalable as cells grow in suspension, does not require animal-derived products or the use of an exogenous virus (adenovirus or baculovirus) and yields genomic titers equal to those obtained in adherent HEK 293 cells in the presence of serum. Most importantly, work load and cost could be dramatically reduced in comparison to earlier methods, when comparing the production of equivalent volumes of cell culture media. Thus, our protocol should appeal to both basic research laboratories and cGMP manufacturing units.
基于腺相关病毒（AAV）的载体是临床研究中最具前景的基因转移载体之一。为了给后期临床试验以及潜在的商业化阶段提供载体，需要一个可扩展的、符合cGMP标准的生产流程。目前几乎所有在I期临床试验中获批的载体生产方案都依赖于在有血清存在的情况下，在贴壁的HEK 293细胞中生产AAV。在本研究中，我们提出了一种在适应悬浮培养的HEK 293细胞中无辅助病毒和无血清生产AAV载体的方法。该方法基于用25 kDa线性聚乙烯亚胺进行质粒转染。与现有方法相比，我们的系统具有高度可扩展性，因为细胞在悬浮状态下生长，不需要动物源产品或使用外源病毒（腺病毒或杆状病毒），并且产生的基因组滴度与在有血清存在的贴壁HEK 293细胞中获得的滴度相当。最重要的是，在比较生产等量体积的细胞培养基时，与早期方法相比，工作量和成本可以大幅降低。因此，我们的方案应该会吸引基础研究实验室和cGMP生产单位。
