Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease

Mol Ther. 2003 Jul;8(1):29-41. doi: 10.1016/s1525-0016(03)00142-4.


Clinical trials evaluating the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy system for the control of graft-versus-host disease (GVHD) have been limited by low transduction efficiencies and inefficient selection procedures. In this study, we designed and evaluated a novel chimeric suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full-length HSV-tk (DeltaCD34-tk). High-efficiency transfer of DeltaCD34-tk to primary human T cells was accomplished after a single exposure to VSV-G-pseudotyped, Moloney murine leukemia virus-based retrovirus 48 h after activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Using an optimized 5-day transduction and selection procedure, transduction efficiencies averaged 71%, with isolation purities greater than 95% and yields exceeding 90%. The immunoselected T cells were selectively eliminated by GCV (IC(50) approximately 3 nM), maintained a normal subset composition, exhibited a polyclonal TCR Vbeta family repertoire, and contained 5 or 6 vector copies per transduced cell when optimally transduced. No increase in GCV sensitivity was observed upon incorporation of highly active mutant HSV-tk enzymes into the DeltaCD34-tk suicide gene. T cells modified with the DeltaCD34-tk gene using the optimized protocol should improve the overall efficacy of the HSV-tk/GCV suicide gene therapy method of GVHD control.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antigens, CD34 / biosynthesis*
  • CD28 Antigens / biosynthesis
  • CD3 Complex / biosynthesis
  • Cell Line
  • Dose-Response Relationship, Drug
  • Flow Cytometry
  • Gene Transfer Techniques
  • Genetic Therapy / methods*
  • Genetic Vectors
  • Graft vs Host Disease / therapy*
  • Humans
  • Inhibitory Concentration 50
  • Leukocytes, Mononuclear / metabolism
  • Magnetics
  • Mice
  • Models, Genetic
  • Mutation
  • NIH 3T3 Cells
  • Protein Structure, Tertiary
  • Retroviridae / genetics
  • Reverse Transcriptase Polymerase Chain Reaction
  • T-Lymphocytes / metabolism*
  • Time Factors
  • Transfection


  • Antigens, CD34
  • CD28 Antigens
  • CD3 Complex