title: Development of a competitive PCR method for in vitro and in vivo quantification of herpes simplex virus thymidine kinase and neomycin resistance-expressing cells used in a clinical trial.
abstract: The aim of this study was to set up a sensitive and specific method to quantify the number of gene-modified cells in a gene therapy clinical trial currently underway at our institution. This trial involves the use of retrovirally transduced allogeneic T cells expressing the herpes simplex-1 thymidine kinase (HSV-TK) and neomycin-phosphotransferase (NeoR) resistance gene. Quantification by competitive PCR was performed, with two homologous internal standards (deltaTK, deltaNeoR), 30 bp shorter than the target sequences (TK, NeoR), coupled to fluorescent laser-based detection. Assessment of the amplification systems procedures was carried out for each sequence. The 30-bp deletion did not affect the amplification efficiency significantly. Determination of the plateau phase of both amplified sequences demonstrated that each sample must be quantified during the predetermined exponential phase. Finally, a blinded study of a transduced cell dilutions panel validated the overall methodology. The competitive PCR was applied to quantification of the retroviral transduction process by quantifying the NeoR gene in transduced PBMC samples (prior to G418 selection) from 18 donors in our clinical trial. A mean transduction efficiency of 9.78% +/- 1.37% was observed. We also quantified TK-expressing donor transgenic T cells in a murine GvHD model. Results demonstrated on initial expansion of donor HSV-TK- expression T cells as well as a significant ganciclovir (GCV)-induced decrease correlated with the number of circulating gene-modified T cells. Therefore, we have developed an efficient gene quantification tool that should be useful for in vivo monitoring of gene-modified cells.
authors.affiliation: Laboratoire de Thérapeutique Immuno-Moléculaire, ETS de Franche Comté, Besançon, France.
authors.forename: S
authors.initials: S
authors.lastname: Maddens
authors.forename: P
authors.initials: P
authors.lastname: Tiberghien
authors.forename: E
authors.initials: E
authors.lastname: Contassot
authors.forename: J M
authors.initials: JM
authors.lastname: Certoux
authors.forename: D
authors.initials: D
authors.lastname: Chalmers
authors.forename: E
authors.initials: E
authors.lastname: Otto
authors.forename: P
authors.initials: P
authors.lastname: Hervé
authors.forename: C
authors.initials: C
authors.lastname: Ferrand
mesh_terms: D000818:Animals; D002986:Clinical Trials as Topic; D004274:DNA, Recombinant / Q000097:blood; D004195:Disease Models, Animal; D004352:Drug Resistance, Microbial / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D006086:Graft vs Host Disease / Q000097:blood / Q000235:genetics; D006801:Humans; D051379:Mice; D009355:Neomycin; D016133:Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D012680:Sensitivity and Specificity; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000637:transplantation; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection; D014184:Transplantation, Homologous / Q000379:methods; D014185:Transplantation, Isogeneic / Q000379:methods
chemical_list: D004274:DNA, Recombinant; D013937:Thymidine Kinase; D009355:Neomycin