PACKAGING CELLS BASED ON INDUCIBLE GENE AMPLIFICATION FOR THE PRODUCTION OF ADENOASSOCIATED VIRUS VECTORS

Although vectors based on adeno-associated virus (AAV) offer several u nique advantages, their usage has been hampered by the difficulties en countered in vector production. In this report, we describe a new AAV packaging system based on inducible amplification of integrated helper and vector constructs containing the simian virus 40 (SV40) replicati on origin. The packaging and producer cell lines developed express SV4 0 T antigen under the control of the reverse tetracycline transactivat or system, which allows inducible amplification of chromosomal loci li nked to the SV40 origin. Culturing these cells in the presence of doxy cycline followed by adenovirus infection resulted in helper and vector gene amplification as,fell as higher vector titers. Clonal producer c ell lines generated vector titers that were 10 times higher than those obtained by standard methods, with approximately 10(4) vector particl es produced per cell, These stocks were free of detectable replication -competent virus. The lack of a transfection step combined with the re producibility of stable producer lines makes this packaging method ide ally suited for the large-scale production of vector stocks for human gene therapy.