Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media

Abstract

Several patients with severe combined immunodeficiency-X1 disease and adenosine deaminase deficiency have been cured by retroviral-mediated gene therapy. Despite the earlier success, the production of retroviral vectors for clinical gene therapy is cumbersome, costly and lacks safety features because of the adherent nature of packaging cells and the necessity to supplement the culture media with bovine serum. The aim of this study was to generate a retrovirus packaging cell line that could be used for the production of large clinical batch vectors. Bicistronic vectors containing an internal ribosomal entry site followed by a selection gene were used to express Moloney murine leukemia gag-pol and amphotropic envelope viral proteins in HEK293 cells. The candidate clone (293GP-A2) that was selected as the packaging cell line could release recombinant green fluorescent protein retroviruses at 4 Â 10 7 infectious viral particles per ml. Similar titers were achieved after these cells were adapted to grow in suspension and serum-free media. Furthermore, using the same culture conditions viral titers proved to be stable for a 3-month culture period. The 293GP-A2 packaging cell line has the potential to be cultured in bioreactors, opening the possibility for large-scale use of retroviral vectors in late stage clinical trials.

Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

“…These cells are safer and release recombinant retroviruses with higher titers compared with former versions (Cosset et al, 1995;Sheridan et al, 2000). The GFP viruses used in this study were produced with a set of 293-based retrovirus packaging cell lines, allowing for the production of vectors with titers above 10 7 ivp/mL (Ghani et al, 2007;Ghani et al, 2009). The transduction ability of GFP vectors pseudotyped with the Ampho, Baev, Galv and RD114 envelopes was assessed.…”

“…HT-1080 cells, 293T cells, the retrovirus parental packaging cell lines 293Vec-Ampho, 293Vec-Baev, www.ecmjournal.org 293Vec-Galv, 293Vec-RD114 (Ghani et al, 2007;Ghani et al, 2009) Human fibroblasts (18-and 37-year-old donors) and keratinocytes (26-and 37-year-old donors) were obtained from reductive breast surgery of healthy adult women after informed consent was given. Fibroblasts and keratinocytes were isolated by the two-step thermolysine and trypsin method, as previously described Lavoie et al, 2013).…”

“…The use of high-titer retroviral packaging cell lines (293Vec cells), constructed in our laboratory, could circumvent the titer issue. They release viral particles pseudotyped with the amphotropic (Ampho), the baboon endogenous virus (Baev), the gibbon ape leukaemia virus (Galv) and the feline endogenous retrovirus (RD114) envelopes (Ghani et al, 2007;Ghani et al, 2009). In the present study, the potential of GFP and COL7A1 vectors, produced from these packaging cell lines, for the transduction of human keratinocytes and fibroblasts was investigated.…”

Concepts for the Production of Viruses and Viral Vectors in Cell Cultures

“…The packaging cell lines thus continuously release RNA-containing vector particles into the medium. Established packaging cell lines can produce retroviral particles over a long period depending on their growth characteristics and are suitable for pilot-scale processes with a bioreactor volume of up to 50 L [14,15]. [20], making them an effective tool for gene therapy.…”

Production of Retroviral and Lentiviral Gene Therapy Vectors: Challenges in the Manufacturing of Lipid Enveloped Virus

“…After CEMFLYA, other high-titer suspension cells were reported, namely suspension-adapted 293GPG cells producing MLV retrovirus vector pseudotyped with the vesicular stomatitis virus G (VSVG) envelope protein and expressing a TK-GFP fusion protein in a 3L acoustic filter-based perfusion bioreactor (Ghani et al 2006). Another major landmark was achieved when the same group published for the first time retroviral vector production in suspension and under serum-free conditions (Ghani et al 2007) (see section 3.4.1). Following retrovirus, lentiviral vector manufacture using suspension cultures has also been recently reported both for transfection-based transient production (Ansorge et al 2009), as well as, for stable production using (inducible) packaging cell lines (Broussau et al 2008).…”