Abnormal expression of only the CD34 part of a transgenic CD34/herpes simplex virus-thymidine kinase fusion protein is associated with ganciclovir resistance.

TY - JOUR

T1 - Abnormal expression of only the CD34 part of a transgenic CD34/herpes simplex virus-thymidine kinase fusion protein is associated with ganciclovir resistance.

AU - Bennour, Emad

AU - Ferrand, Christophe

AU - Rémy-Martin, Jean-Paul

AU - Certoux, Jean-Marie

AU - Gorke, Sebastian

AU - Qasim, Waseem

AU - Gaspar, H Bobby

AU - Baumert, Thomas

AU - Duperrier, Anne

AU - Deschamps, Marina

AU - Fehse, Boris

AU - Tiberghien, Pierre

AU - Robinet, Eric

PY - 2008

Y1 - 2008

N2 - Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.

AB - Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.

M3 - SCORING: Zeitschriftenaufsatz

VL - 19

SP - 699

EP - 709

JO - HUM GENE THER

JF - HUM GENE THER

SN - 1043-0342

IS - 7

M1 - 7

ER -