Mezzina Mauro, Merten Otto-Wilhelm
Généthon, Evry, France.
Methods Mol Biol. 2011;737:211-34. doi: 10.1007/978-1-61779-095-9_9.
Adeno-associated virus (AAV) vectors have evolved over the past decade as a particularly useful gene -vector for in vivo applications. In contrast to oncoretro- and lentiviral vectors, this vector stays essentially episomal after gene transfer, making it safer because of the absence of insertional mutagenesis. AAV's non-pathogenicity is a further advantage. For decades, this vector could only be produced at a small scale for research purposes and, eventually, used at very small doses for clinical studies, because only transfection methods were available, which have limited scalability. However, since the development of scalable production methods, this bottleneck is resolved and, from a technical point of view, large quantities of AAV vectors can be produced, opening the possibility of using AAV vectors for whole body treatments in gene therapy trials. This chapter presents the basic principles of small- and large-scale production procedures as well as detailed procedure of small-scale production, purification, and analytical protocols for AAV vectors. In Chapter 10, the reader will find a large-scale production method based on the use of the insect cell/baculovirus system.
在过去十年中，腺相关病毒（AAV）载体已发展成为一种特别适用于体内应用的基因载体。与致癌逆转录病毒载体和慢病毒载体不同，这种载体在基因转移后基本上保持游离状态，由于不存在插入诱变，因此更安全。AAV的无致病性是另一个优势。几十年来，这种载体只能小规模生产用于研究目的，最终在临床研究中以非常小的剂量使用，因为当时只有转染方法可用，而转染方法的可扩展性有限。然而，自从开发出可扩展的生产方法以来，这一瓶颈已得到解决，从技术角度来看，可以大量生产AAV载体，这为在基因治疗试验中使用AAV载体进行全身治疗开辟了可能性。本章介绍了AAV载体小规模和大规模生产程序的基本原理，以及小规模生产、纯化和分析方案的详细程序。在第10章中，读者将找到一种基于昆虫细胞/杆状病毒系统的大规模生产方法。
