Episomal Vectors Rapidly and Stably Produce High-Titer Recombinant Retrovirus

Abstract

The nuclear replication and retention functions of the Epstein–Barr virus (EBV) have been utilized here to maintain retroviral constructs episomally within human cell-based retroviral packaging lines. These hybrid EBV/retroviral constructs are capable of producing helper-free recombinant retrovirus as soon as 48 hr and for at least 30 days after transfection into 293T-based ecotropic and/or amphotropic retroviral packaging cells. Viral titers greater than 107 TU/ml were obtained after puromycin selection of transfected retroviral packaging cells. This episomal approach to retroviral production circumvents some limitations inherent in transient and chromosomally stable retroviral producer systems, affording reproducibly rapid, large-scale, stable, and high-titer retrovirus production. Producing high-titer retrovirus can be a limiting factor in the successful application of retroviruses for gene transduction. A retroviral producer system capable of both rapid and long-term virus production would circumvent many of the restrictions inherent to chromosomal-stable and/or transient-based production methods. It is demonstrated here that replication and nuclear retention functions of the human Epstein–Barr virus (EBV) can confer stable episomal maintenance to retroviral constructs within human cell-based packaging lines. Such hybrid EBV/retroviral constructs permit both rapid and long-term production of high-titer retrovirus. Furthermore, it is demonstrated that these constructs are not prone to arrangements when maintained episomally in packaging cells and that viral stocks are helper-virus free.