Molecular mechanism for ganciclovir resistance in human T lymphocytes transduced with retroviral vectors carrying the herpes simplex virus thymidine kinase gene.
The herpes simplex virus thymidine kinase gene type 1 (HSV-Tk) ganciclovir (GCV) system is a novel therapeutic strategy for the modulation of graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation (allo-SCT). Retroviral-mediated gene transfer of the HSV-Tk gene into donor T lymphocytes before allo-SCT may allow their in vivo selective depletion after treatment with GCV. The expression of the HSV-Tk gene was analyzed in vitro in CEM cells, a human lymphoblastoid cell line, transduced with 2 different vectors, each containing the HSV-Tk gene and a selectable marker gene. GCV-resistant clones were identified within the clones expressing the marker gene. Characterization of the molecular events leading to this resistance revealed a 227-bp deletion in the HSV-Tk gene due to the presence of cryptic splice donor and acceptor sites within the HSV-Tk gene sequence. Furthermore, it was confirmed that this deletion was present in human primary T cells transduced with either vector and in 12 patients who received transduced donor T cells, together with a T-cell-depleted allo-SCT. In vivo circulating transduced T cells containing the truncated HSV-Tk gene were identified in all patients immediately after infusion and up to 800 days after transplantation. In patients who received GCV as treatment for GVHD, a progressive increase in the proportion of transduced donor T cells carrying the deleted HSV-Tk gene was observed. These results suggest that the limitations within the HSV-Tk/GCV system can be improved by developing optimized retroviral vectors to ensure maximal killing of HSV-Tk-transduced cells.
Garin
M I
MI
Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom.
Garrett
E
E
Tiberghien
P
P
Apperley
J F
JF
Chalmers
D
D
Melo
J V
JV
Ferrand
C
C
D000998:Antiviral Agents / Q000008:administration & dosage / Q000494:pharmacology; D018929:Cell Culture Techniques; D002460:Cell Line; D004351:Drug Resistance; D015774:Ganciclovir / Q000008:administration & dosage / Q000494:pharmacology*; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000494:pharmacology; D006086:Graft vs Host Disease / Q000628:therapy; D019337:Hematologic Neoplasms / Q000628:therapy; D006801:Humans; D008212:Lymphocyte Depletion / Q000379:methods; D012190:Retroviridae / Q000235:genetics; D017422:Sequence Analysis, DNA; D018139:Simplexvirus / Q000201:enzymology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000637:transplantation* / Q000821:virology; D013937:Thymidine Kinase / Q000235:genetics / Q000627:therapeutic use*; D014161:Transduction, Genetic
D000998:Antiviral Agents; D013937:Thymidine Kinase; D015774:Ganciclovir