Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease.
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.
Rettig
Michael P
MP
Division of Oncology, Siteman Cancer Center, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Ritchey
Julie K
JK
Meyerrose
Todd E
TE
Haug
Jeffrey S
JS
DiPersio
John F
JF
D000818:Animals; D018952:Antigens, CD34 / Q000096:biosynthesis*; D018106:CD28 Antigens / Q000096:biosynthesis; D017252:CD3 Complex / Q000096:biosynthesis; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006086:Graft vs Host Disease / Q000628:therapy*; D006801:Humans; D020128:Inhibitory Concentration 50; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008280:Magnetics; D051379:Mice; D008957:Models, Genetic; D009154:Mutation; D041681:NIH 3T3 Cells; D017434:Protein Structure, Tertiary; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D018952:Antigens, CD34; D018106:CD28 Antigens; D017252:CD3 Complex