Abnormal expression of only the CD34 part of a transgenic CD34/herpes simplex virus-thymidine kinase fusion protein is associated with ganciclovir resistance.
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.
INSERM U645, 25020 Besançon, France.
Emad
E
Bennour
Christophe
C
Ferrand
Jean-Paul
JP
Rémy-Martin
Jean-Marie
JM
Certoux
Sebastian
S
Gorke
Waseem
W
Qasim
H Bobby
HB
Gaspar
Thomas
T
Baumert
Anne
A
Duperrier
Marina
M
Deschamps
Boris
B
Fehse
Pierre
P
Tiberghien
Eric
E
Robinet
D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism*; D000998:Antiviral Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D024882:Drug Resistance, Viral; D015774:Ganciclovir / Q000494:pharmacology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013601:T-Lymphocytes / Q000276:immunology / Q000821:virology; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes
D018952:Antigens, CD34; D000998:Antiviral Agents; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir