Low-speed centrifugation of retroviral vectors absorbed to a particulate substrate: a highly effective means of enhancing retroviral titre

Abstract

For many gene therapy applications the effective titre of retroviral vectors is a limiting factor both in vitro and in vivo. Purification and concentration of retrovirus from packaging cell supernatant can overcome this problem. To this end we have investigated a novel procedure which involves complexing retrovirus to a dense and particulate substrate followed by a short low-speed centrifugation. The study reported here uses heat-killed, formaldehyde fixed Staphylococcus aureus (Pansorbin) absorbed to PG13 derived retrovirus. This complex was then used to harvest retrovirus from packaging cell supernatant: centrifugation and washing of

Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography

“…Considerable efforts have been made to develop a variety of processes for purification of retroviral vectors. Both ultracentrifugation and low-speed centrifugation have been employed to prepare highly concentrated retroviral vectors (3,8,10,36). Cosedimentation of small cell-derived vesicles, as well as serum proteins, with the viral particles led to even more serious contamination in those concentrated retroviral vectors (4,19).…”

Affinity recovery of Moloney Murine Leukaemia Virus

“…Retroviral particles produced from BiotinSE treated Murine fibroblast-derived PG13 packaging cells (Moloney Murine Leukaemia Virus) acquired biotinylated envelope proteins as they bud from the PG13 cell membrane as described in detail in Hughes et al [8,9]. Briefly, PG13 producer cells were trypsinised and plated at 1 × 10 6 cells/90 mm petridish.…”

“…PG13 pBabe.puro producer cells were generated as previously described in [9]. The cells are NIH 3T3 based retroviral packaging cells with the Gibbon Ape Leukaemia virus envelope gene that produce MoMuLV transferring puromycin resistance.…”

“…During continued culture the budding process associates the biotin modified env with the retroviral core resulting in the production of biotin labelled retrovirus. Furthermore, they demonstrated that the virus retained infectivity whilst still bound to adsorbent particles [8,9]. Streptavidin is a tetrameric protein derived from Streptomyces avidinii that demonstrates a high affinity for biotin (K d = 10 −13 -10 −16 M), binding four moles of biotin per mole of protein with a binding site in each protein subunit [10,11].…”

Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer

“…Unfortunately, retroviruses are often inactivated by this procedure, presumably because shear forces cause the retrovirus envelope proteins to separate from the virus particles, giving rise to virus particles that are no longer fusogenic. Numerous alternatives to ultracentrifugation as a means to purify retrovirus have been developed, including low-speed centrifugation, size-exclusion membrane filtration, affinity chromatography, and complexation with calcium phosphate, cationic polymers, or paramagnetic beads (Bowles et al, 1996;Burns et al, 1993;Coleman et al, 2003;Darling et al, 2000;Gatlin et al, 2001;Hughes et al, 2001;Kuiper et al, 2002;Miller et al, 1996;Paul et al, 1993;Reiser, 2000;Scherr et al, 2002;Yamada et al, 2003;Yang et al, 2002;Ye et al, 2004;Zelenock et al, 1997;Zhang et al, 2001). Although each of these approaches are capable of concentrating retrovirus stocks and improving gene transfer, they do not increase the number of genes transferred to the same extent that the virus stocks are concentrated, which suggests that these virus processing methods co-concentrate inhibitors (Le Doux et al, 1998;Seppen et al, 2000) or directly reduce the ability of the viruses to transduce cells.…”