公开(公告)号：US5436146A

公开(公告)日：1995-07-25

申请号：US7399

申请日：1993-01-21

申请人： Thomas E. Shenk ,  Richard J. Samulski ,  Long-Sheng Chang

发明人： Thomas E. Shenk ,  Richard J. Samulski ,  Long-Sheng Chang

IPC分类号： C12N5/02 ,  C12N7/01 ,  C12N15/00 ,  C12N15/09 ,  C12N15/63 ,  C12N15/70 ,  C12N15/74 ,  C12N15/86 ,  C12N15/864 ,  C12Q1/68 ,  C12Q1/70 ,  G01N33/53 ,  C12N5/10 ,  C12N15/35

CPC分类号： C12N15/86 ,  C12N2750/14143

摘要： The present invention relates to a method for producing helper-free stocks of recombinant adeno-associated virus (rAAV) which can be used to efficiently and stably transduce foreign genes into host cells or organisms. The method comprises the cotransfection of eukaryotic cells with rAAV and with helper AAV DNA in the presence of helper virus (e.g. adenovirus or herpesvirus) such that the helper AAV DNA is not associated with virion formation. The crux of the invention lies in the inability of the helper AAV DNA to recombine with rAAV vector, thereby preventing the generation of wild-type virus. In a specific embodiment of the invention, the vector comprises a recombinant AAV genome containing only the terminal regions of the AAV chromosome bracketing a non-viral gene, and the helper AAV DNA comprises a recombinant AAV genome containing that part of the AAV genome which is not present in the vector, and in which the AAV terminal regions are replaced by adenovirus sequences. In a further embodiment of the invention, cell lines are created which incorporate helper AAV DNA which can directly produce substantially pure recombinant AAV virus. The pure stocks of recombinant AAV produced according to the invention provide an AAV viral expression vector system with increased yield of recombinant virus, improved efficiency, higher definition, and greater safety than presently used systems.