1. ¹Department of Gene Regulation and Differentiation, German Research Center for Biotechnology, D-38124 Braunschweig, Germany
2. ²Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica, Apartado 12, P-2781-901 Oeiras, Portugal
3. ³Généthon, 1 bis, Rue de l'Internationale-BP60, F-91002 Evry Cedex, France
4. ⁴Laboratoire de Vectorologie Retrovirale et Therapie Genique, INSERM, 46 Allee d'Italie, 69364 Lyon Cedex 07, France

Correspondence: Dagmar Wirth, GBF–National Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Germany. Fax: +49 531 6181 262. E-mail: dagmar.wirth@gbf.de

Received 11 November 2005; Revised 9 December 2005; Accepted 13 December 2005.

Abstract

We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 10⁷ infectious particles/10⁶ cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.