Farson Deborah, Harding Thomas C, Tao Lucy, Liu Jun, Powell Sandra, Vimal Vishalini, Yendluri Satyasri, Koprivnikar Kathryn, Ho Kenneth, Twitty Christopher, Husak Paul, Lin Andy, Snyder Richard O, Donahue Brian A
Cell Genesys, Inc., 500 Forbes Blvd., South San Francisco, CA 94080, USA.
J Gene Med. 2004 Dec;6(12):1369-81. doi: 10.1002/jgm.622.
One of the major limitations to the use of adeno-associated virus (AAV) vectors for gene therapy has been the difficulty in producing enough vector to supply a clinical trial. More than 20 000 roller bottles may be required to generate AAV by the traditional transient transfection process to treat 50 patients. A scalable AAV producer cell line grown in serum-free media will meet the needs for the manufacture of AAV gene therapeutics.
A packaging cell line was generated by introducing the AAV rep and cap genes into A549 cells. From this packaging cell line, a number of producer cell lines were generated by infecting the packaging cell with the appropriate AAV vector. Producer cell lines were then adapted to serum-free suspension conditions for growth in bioreactors.
We report here the development of six AAV producer cell lines that generate > 10(4) particles/cell. The rAAV vector preparations from these cell lines have physical and functional characteristics similar to rAAV vectors prepared by transient transfection. To enable large-scale production, producer cell lines were adapted to serum-free suspension and we demonstrate production of AAV at the 15 L scale. In addition, vector preparations from these cell lines were shown to be free of wild-type AAV.
AAV producer cell lines can be readily scaled to meet the needs of clinical trials. One 500 L bioreactor of these producer cells can produce the equivalent of 2500 high capacity roller bottles or 25 000 T-175 tissue culture flasks.
腺相关病毒（AAV）载体用于基因治疗的主要限制之一是难以生产足够的载体以供应临床试验。通过传统的瞬时转染过程生产用于治疗50名患者的AAV可能需要超过20000个滚瓶。在无血清培养基中生长的可扩展AAV生产细胞系将满足AAV基因治疗药物生产的需求。
通过将AAV的rep和cap基因导入A549细胞来构建包装细胞系。从该包装细胞系中，通过用合适的AAV载体感染包装细胞产生了多个生产细胞系。然后使生产细胞系适应无血清悬浮培养条件以便在生物反应器中生长。
我们在此报告了六种AAV生产细胞系的开发，这些细胞系每个细胞可产生>10⁴个病毒颗粒。来自这些细胞系的重组AAV载体制剂具有与通过瞬时转染制备的重组AAV载体相似的物理和功能特性。为了实现大规模生产，使生产细胞系适应无血清悬浮培养，并且我们展示了在15L规模下AAV的生产。此外，来自这些细胞系的载体制剂显示不含野生型AAV。
AAV生产细胞系可以很容易地进行扩大培养以满足临床试验的需求。一个500L生物反应器的这些生产细胞所产生的AAV相当于2500个大容量滚瓶或25000个T-175组织培养瓶所产生的量。
