公开(公告)号：US20050266567A1

公开(公告)日：2005-12-01

申请号：US11170269

申请日：2005-06-28

申请人： Edward Atkinson ,  Victor Fung ,  Perry Wilkins ,  Ryan Takeya ,  Thomas Reynolds ,  Ian Aranha

发明人： Edward Atkinson ,  Victor Fung ,  Perry Wilkins ,  Ryan Takeya ,  Thomas Reynolds ,  Ian Aranha

IPC分类号： A61K48/00 ,  C07C2/32 ,  C07D207/32 ,  C07D207/34 ,  C07D207/50 ,  C07D295/30 ,  C07D401/14 ,  C07D409/14 ,  C07F7/08 ,  C07F9/572 ,  C07F9/58 ,  C07F15/04 ,  C07K14/015 ,  C08F4/659 ,  C08F10/00 ,  C08F110/02 ,  C08F110/14 ,  C08F210/16 ,  C12N5/10 ,  C12N7/00 ,  C12N7/02 ,  C12N15/861 ,  C12N15/864

CPC分类号： C07D207/32 ,  A61K48/0091 ,  C07C2/32 ,  C07C2531/22 ,  C07D207/34 ,  C07D207/50 ,  C07D295/30 ,  C07D401/14 ,  C07D409/14 ,  C07F7/0812 ,  C07F9/572 ,  C07F9/58 ,  C07F15/045 ,  C07K14/005 ,  C12N7/00 ,  C12N15/86 ,  C12N2750/14122 ,  C12N2750/14143 ,  C12N2750/14151 ,  C12N2750/14152

摘要： This invention provides methods and compositions for producing high titer, substantially purified preparations of recombinant adeno-associated virus (AAV) that can be used as vectors for gene delivery. At the onset of vector production, AAV producer cells of this invention typically comprise one or more AAV packaging genes, an AAV vector comprising a heterologous (i.e. non-AAV) transgene of interest, and a helper virus such as an adenovirus. The AAV vector preparations produced are generally replication incompetent but are capable of mediating delivery of a transgene of interest (such as a therapeutic gene) to any of a wide variety of tissues and cells. The AAV vector preparations produced according to this invention are also substantially free of helper virus as well as helper viral and cellular proteins and other contaminants. The invention described herein provides methods of producing rAAV particles by culturing producer cells under conditions, such as temperature and pH, that promote release of virus. Also provided is a quantitative, high-throughput assay useful in the assessment of viral infectivity and replication, as well as in the screening of agent that affect viral infectivity and/or replication.

摘要翻译： 本发明提供用于生产可用作基因递送载体的重组腺相关病毒（AAV）的高滴度，基本纯化的制剂的方法和组合物。 在载体生产开始时，本发明的AAV生产细胞通常包含一个或多个AAV包装基因，包含感兴趣的异源（即非AAV）转基因的AAV载体和辅助病毒如腺病毒。 产生的AAV载体制剂通常是复制无能力的，但能够介导感兴趣的转基因（例如治疗基因）到多种组织和细胞中的任何一种。 根据本发明制备的AAV载体制剂也基本上不含辅助病毒以及辅助病毒和细胞蛋白质和其它污染物。 本文描述的本发明提供了通过在促进病毒释放的条件例如温度和pH下培养生产细胞来生产rAAV颗粒的方法。 还提供了用于评估病毒感染性和复制以及影响病毒感染性和/或复制的试剂筛选的定量，高通量测定。