Efficient lentiviral transduction of primary mouse T cells
Year: 2008
Session type: Poster / e-Poster / Silent Theatre session
1University of Manchester, Manchester, UK, 2Paterson Institute for Cancer Research, Manchester, UK
Abstract
Unlike Human T cells, Mouse T-cells have proven to be resistant to HIV at multiple levels of the infection cycle [1, 2]. In order to explore if this precludes the use of HIV derived lentiviral gene therapy vectors for mouse T-cell transduction, splenic T cells were mixed with eGFP encoding 3rd generation lentiviral vectors [3] at a m.o.i of 10:1 and polyclonally activated using antibody coated beads. After five days of culture, eGFP transduced T cells were clearly identified. However, the choice of promoter proved important with the rank order of potency of promoters as follows: human PGK > human EF1α > SFFV > CMV. Expression was maintained for at least one month after adoptive transfer of low numbers of gene-modified T cells into conditioned mice. Importantly, a retroviral vector pseudotyped with the VSV-g envelope failed to efficiently transduce mouse T cells when similarly added to the T cells at the start of the activation process. The lentiviral vectors used included a human PRE region; however, replacing this region with the woodchuck PRE did not abolish primary mouse T cell transduction. Furthermore, primary splenic T cells cultured in the absence of activatory antibodies were also transduced although this was dependent upon the presence of the common γ cytokines IL-2 and IL-7. Given the correct choice of promoter, primary mouse T cells are suitable targets to explore lentiviral gene therapy approaches.
References
[1] J Virol. 2007 Jan; 81(2):677-88
[2] J Virol. 2004 Nov; 78(22):12537-47
[3] J Hepatol. 2002 Apr; 36(4):459-65
Unlike Human T cells, Mouse T-cells have proven to be resistant to HIV at multiple levels of the infection cycle [1, 2]. In order to explore if this precludes the use of HIV derived lentiviral gene therapy vectors for mouse T-cell transduction, splenic T cells were mixed with eGFP encoding 3rd generation lentiviral vectors [3] at a m.o.i of 10:1 and polyclonally activated using antibody coated beads. After five days of culture, eGFP transduced T cells were clearly identified. However, the choice of promoter proved important with the rank order of potency of promoters as follows: human PGK > human EF1α > SFFV > CMV. Expression was maintained for at least one month after adoptive transfer of low numbers of gene-modified T cells into conditioned mice. Importantly, a retroviral vector pseudotyped with the VSV-g envelope failed to efficiently transduce mouse T cells when similarly added to the T cells at the start of the activation process. The lentiviral vectors used included a human PRE region; however, replacing this region with the woodchuck PRE did not abolish primary mouse T cell transduction. Furthermore, primary splenic T cells cultured in the absence of activatory antibodies were also transduced although this was dependent upon the presence of the common γ cytokines IL-2 and IL-7. Given the correct choice of promoter, primary mouse T cells are suitable targets to explore lentiviral gene therapy approaches.
References
[1] J Virol. 2007 Jan; 81(2):677-88
[2] J Virol. 2004 Nov; 78(22):12537-47
[3] J Hepatol. 2002 Apr; 36(4):459-65
