Robust Enhancement of Lentivirus Production by Promoter Activation

Abstract

Lentiviral vectors are a valuable tool to deliver exogenous genes for stable expression in cells. While much progress has been made in processing lentiviral vector-containing culture medium, it remains to be explored how the production of lentiviral vector from producer cells can be increased. We initially found that co-expression of the SPRY domain-containing SOCS box protein 1 (SPSB1) promotes the production of human immunodeficiency virus type 1 (HIV-1) and lentiviral vector with increased expression of the Gag and envelope proteins and activation of the HIV-1 LTR and CMV promoter. The presence of AP-1, NF-κB and CREB/ATF recognition sites in these promoters prompted us to utilize human T-lymphotropic virus type 1 (HTLV-1) Tax for lentiviral vector production because Tax activates all these transcription factors. Co-expression of a small amount of Tax markedly increased both the expression of viral structural proteins in producer cells and release of lentiviral vector particles, resulting in a more than 10-fold enhancement of transduction efficiency. Of note, the Tax protein was not detected in the lentiviral vector particles concentrated by ultracentrifugation, supporting the safety of this preparation. Collectively, these results indicate that promoter activation in producer cells represents a promising approach to preparing high-titer lentiviral vectors.

Implementation of Novel Affinity Ligand for Lentiviral Vector Purification

“…In the flow-through, no signal was detected showing that the Lenti VSVG Affinity Matrix could successfully capture the VSVG-LVs particles without resin saturation. In contrast, a strong signal was found in the elution fraction, which exhibited the characteristic LVs p24 pattern [ 61 , 62 ], supporting that an efficient viral elution occurred. For the strip sample, a subtle signal was detected, although it was stronger for the p24 protein.…”

A lentiviral vector for the production of T cells with an inducible transgene and a constitutively expressed tumour-targeting receptor

“…Article https://doi.org/10.1038/s41551-023-01013-5 enhancer, and showed that we could increase viral titres in the presence of Tax 65 , and to a greater extent when combined with NovB2.…”

Assessment and streamlined preparation of low-cytotoxicity lentiviral vectors for mobilized human hematopoietic stem cell transduction

“…By use of c92 cells in perfusion mode bioreactors (and with multiple harvesting), 8 £ 10 10 transduction units can be generated per liter. In an alternate approach, ectopic expression of HTLV-1 −derived tax, an enhancer for several common transcription factors (e.g.., AP1, ATF, NFKB) [28], has recently been reported to boost lentivirus production in 293-T cells up to 10-fold [29]. For lentiviruses prepared using these progressive systems, the present findings underscore the importance of critically assessing possible cytotoxic activity for such large-scale virus preparations; the load of possibly damaged and inactive virus; and the effectiveness of these viruses in efficiently transducing refractive primary cell populations.…”