Improvement of Retroviral Packaging Cell Lines by Introducing the Polyomavirus Early Region

Abstract

To obtain high-titer recombinant retroviruses, we constructed plasmid pDL⁺, which carries the extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Although pDL⁺ is useful for obtaining high-titer recombinant retroviruses, this vector plasmid is difficult to modify further for tissue-specific expression of foreign genes. To overcome this problem, the coding region of the polyomavirus early gene was expressed in the packaging cell lines. We modified the packaging cell lines, Ψ2 and PA317, by stably introducing the polyomavirus early gene, and established ΨMP34 and ΨMP37 from Ψ2, and PAMP51 from PA317. In the transient expression system using plasmids with and without the polyomavirus replication origin, the titers of recombinant retrovirus produced by these cell lines were 10–100 times higher than produced by the parent cell line and reached levels of 0.5–1.5 × 10⁶ cfu/ml. Expression of the polyomavirus early gene in the packaging cell lines did not stimulate the production of replication-competent retrovirus. We also routinely established stable clones producing retrovirus titers over 1 × 10⁷ cfu/ml from ΨMP34 and PAMP51. We found that the activity of the long terminal repeat (LTR) promoter is stimulated by the polyomavirus early region protein(s) in these cell lines. Therefore, increases titer can be expected to occur in all the retroviral vectors in which LTR promoter is used to transcribe the retroviral genome. Although recombinant retroviruses have been widely used for gene transfer to the target cells, their low titers are one of the disadvantages of retroviral vectors. To achieve higher viral titers, the structure of the vector plasmids and the system for their preparation were improved. In this study, we improved the packaging cell lines by introducing the polyomavirus early region. Expression of the polyomavirus early gene in the packaging cell lines enhances long terminal repeat promoter activity and causes high production of recombinant retrovirus.