Ganciclovir mediated killing of t cells transduced with vsv-g pseudotyped lentiviral vector expressing HSVTK

Sudhanshu P. Raikwar, Kenneth Cornetta

Research output: Contribution to journalArticlepeer-review


Graft versus host disease (GVHD) is a major complication following allogeneic bone marrow transplantation. A promising new approach to prevent GVHD involves retroviral vector mediated selective gene transfer of donor lymphocytes with herpes simplex virus thymidine kinase (HSVTK). One of the major limitations to this approach is the inherent inability of retroviral vectors to transduce nondividing cells. As an alternative we have explored the potential of lentiviral vectors to transfer the HSVTK gene. Vesicular stomatitis virus (VSV-G) envelope pseudotyped lentiviral vectors expressing GFP (vHRCMVGFP/ VSV) and HSVTK (vHRCMVTK/VSV) were generated by transient transfection of the packaging, envelope and transfer vectors in 293T cells (Naidini et al. Science 272:263-267. 1996). The vector tiler for vHRCMVGFP/VSV was determined by FACS analysis of transduced 293T cells and was found to be 4.8×105 transduction units per mi. Jurkat cells were transduced with vHRCMVGFP/VSV and vHRCMVTK/VSV vectors in the presence of recombinant fibronectin fragment CH-296 (Takara Shuzo). The percentage of GFP expressing Jurkat cells was analyzed by FACS, 3 and 20 days post transduction. GFP expressing cells were 70% on day 3 and 69% on day 20, respectively. Jurkat cells transduced with vHRCMVTK/VSV were treated with Ganciclovir (GCV) concentrations ranging from 5mm to 500mm and the cell viability was determined by MTS assay after 7 days. Untransduced Jurkat cells exhibited an IC50 of 182.4 mM while vHRCMVTK/VSV transduced Jurkat cells revealed an IC50 of 47.5 mm GCV. Jurkat cells transduced with vHRCMVTK/VSV were allowed to proliferate further in culture without any GCV for 20 days and then subjected to GCV treatment. These lentivirus transduced cells exhibited an IC50 of 53.2 mM GCV following an in-vitro expansion for 20 days. Optimization of primary T cell gene transfer is currently ongoing. Our results demonstrate the potential implications of lentiviral vectors for developing novel suicide gene transfer strategies to prevent GVHD.

Original languageEnglish (US)
Pages (from-to)383b
Issue number11 PART II
StatePublished - Dec 1 2000

ASJC Scopus subject areas

  • Biochemistry
  • Immunology
  • Hematology
  • Cell Biology

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