NeoR-based transduced T lymphocytes detected by real-time quantitative polymerase chain reaction.
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.
Venditti
Gigliola
G
Department of Clinical and Experimental Medicine, Haematology and Clinical Immunology Section, University of Perugia, Perugia, Italy 60122.
Di Ianni
Mauro
M
Falzetti
Franca
F
Moretti
Lorenzo
L
Di Florio
Sabrina
S
Tabilio
Antonio
A
D019008:Drug Resistance, Neoplasm; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D016014:Linear Models; D009355:Neomycin / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D018139:Simplexvirus / Q000201:enzymology; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics; D013997:Time Factors; D020298:U937 Cells
D011500:Protein Synthesis Inhibitors; D013937:Thymidine Kinase; D009355:Neomycin