ABSTRACT
To develop a trial with lymphocyte suicide gene therapy in patients with hematological malignancies, we transduced human T lymphocytes with a retroviral vector (LSN-tk) encoding the herpes simplex virus thymidine kinase (tk) and the neomycin resistance (NeoR) genes. Precise quantification of gene transfer is crucial for any gene therapy protocol, but methods using semiquantitative PCR are inaccurate and subject to variations. Real-time quantitative PCR could be a valid alternative. A TaqMan probe was designed to hybridize with the NeoR gene. The PCR product is 64 nucleotides long and readily quantified by TaqMan probe binding. The analysis was performed soon after transduction and repeated after the selection procedure. This method was more accurate, reproducible, and sensitive than the semiquantitative PCR assay. Accuracy was the same whether the analysis was performed at the highest rate or at the lowest rate of transduction. Additionally we used real-time PCR to monitor the kinetics of enrichment of the transduced cells over the selection time and showed how 7 days of selection are needed. This study precisely quantified the percentages of cells transduced by the retroviral vector and could have major implications in gene therapy studies.
Subject(s)
Drug Resistance, Neoplasm , Neomycin/pharmacology , T-Lymphocytes/cytology , Gene Transfer Techniques , Genetic Therapy/methods , Genetic Vectors , Humans , Kinetics , Linear Models , Protein Synthesis Inhibitors/pharmacology , Reproducibility of Results , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Simplexvirus/enzymology , T-Lymphocytes/metabolism , Thymidine Kinase/genetics , Time Factors , U937 CellsABSTRACT
The herpes simplex virus thymidine kinase (HSV-tk) gene conferring ganciclovir (GCV)-specific sensitivity to transduced cells might control Graft-versus-Leukemia (GvL)/Graft-versus-Host Disease (GvHD). Human T lymphocytes were engineered with an LSN-tk retroviral vector encoding tk and neomycin resistance (NeoR) genes. A total of 80 x 10(6) tk(+) lymphocytes were injected intraperitoneally in NOD-SCID mice. Engraftment was evaluated by human CD45(+)/CD3(+) cytofluorimetric analysis and NeoR-based polymerase chain reaction (PCR) on peripheral blood, bone marrow, liver, thymus, and spleen on day +5. After 14 days, GCV (10 mg/kg daily) cytofluorimetric analysis and PCR were repeated (day +19). Immunohistological studies with anti-CD3 monoclonal antibody followed by alkaline phosphatase and monoclonal anti-alkaline phosphatase staining were performed on spleen and liver at the same time points. Human CD45(+)/CD3(+) cells were engrafted in all tissues on day +5 according to cytofluorimetry, immunohistology, and PCR. Lymphocytes "homed" to the white pulp T-cell area and to the red pulp; liver localization is prevalently at the periportal area. After GCV (day +19), cytofluorimetry and immunohistology showed very few CD3(+) cells. PCR identified the transgene in 22% tissue samples (positive only in thymus and spleen). GvHD did not occur in any animal. These data demonstrate elevated doses of human-transduced CD3(+) cells engraft in NOD/SCID mice; after GCV, very few CD3(+) cells can be detected and those that escape treatment can be found in the thymus and in the spleen on day +19. Lack of full response to GCV may account for cases of GvHD in patients receiving tk-transduced T lymphocytes.
