Abnormal expression of only the CD34 part of a transgenic CD34/herpes simplex virus-thymidine kinase fusion protein is associated with ganciclovir resistance.
Fiche publication
Date publication
juillet 2008
Auteurs
Membres identifiés du Cancéropôle Est :
Pr BAUMERT Thomas, Dr FERRAND Christophe
Tous les auteurs :
Bennour E, Ferrand C, Remy-Martin JP, Certoux JM, Gorke S, Qasim W, Gaspar HB, Baumert T, Duperrier A, Deschamps M, Fehse B, Tiberghien P, Robinet E
Lien Pubmed
Résumé
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.
Référence
Hum Gene Ther. 2008 Jul;19(7):699-709.