A New Generation of Retroviral Producer Cells: Predictable and Stable Virus Production by Flp-Mediated Site-Specific Integration of Retroviral Vectors

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 x 10(7) infectious particles/10(6) 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.

Lentivirus Production Is Influenced by SV40 Large T-Antigen and Chromosomal Integration of the Vector in HEK293 Cells

“…It contains Flp recombination targets F-WT and F-5 flanking the viral genome. EMCV-IRES elements and an ATG start codon are positioned upstream to the F-5 site to complement the ATG-deficient neomycin-phosphotransferase expression upon targeting (for details consult Verhoeyen et al, 2001;Schucht et al, 2006).…”

“…It is well known that a critical parameter is the nature of the vector integration site, which defines the expression and thus the titer. This has been studied for single copy vector integration events (Coroadinha et al, 2006;Schucht et al, 2006). Conceptually, safe (i.e., defined and genetically stable) production systems for c-retroviral and lentiviral vectors require a low copy number or, most beneficially, a single vector copy integrated into the producer cell genome.…”

“…c-Retroviral modular producer cell lines have been established that allow production of high titer vectors from single, defined chromosomal sites (Gama-Norton et al, 2010;Coroadinha et al, 2006;Schucht et al, 2006;Loew et al, 2010). This permits production of c-retroviral vectors in a predictable and controlled manner (Coroadinha et al, 2006;Schucht et al, 2006;Loew et al, 2010;Gama-Norton et al, 2010).…”

“…This permits production of c-retroviral vectors in a predictable and controlled manner (Coroadinha et al, 2006;Schucht et al, 2006;Loew et al, 2010;Gama-Norton et al, 2010). However, the development of cellular systems that exploit single chromosomal loci for the production of lentiviral vectors in a predictable and controlled manner has not been achieved so far.…”

Removal of envelope protein‐free retroviral vectors by anion‐exchange chromatography to improve product quality

“…89071904) harboring a MFGSnlsLacZ transgene [18]; HEK 293 derived 1B28F cell line producing amphotropic RVs harboring a pEMCeGFPROL coding for GFP protein [19]. Amphotropic lentiviral vectors were produced by the 293T-derived STAR-A cell line (ECCAC no.…”