title: Measurement of lentiviral vector titre and copy number by cross-species duplex quantitative PCR.
abstract: Lentiviruses are the vectors of choice for many preclinical studies and clinical applications of gene therapy. Accurate measurement of biological vector titre before treatment is a prerequisite for vector dosing, and the calculation of vector integration sites per cell after treatment is as critical to the characterisation of modified cell products as it is to long-term follow-up and the assessment of risk and therapeutic efficiency in patients. These analyses are typically based on quantitative real-time PCR (qPCR), but as yet compromise accuracy and comparability between laboratories and experimental systems, the former by using separate simplex reactions for the detection of endogene and lentiviral sequences and the latter by designing different PCR assays for analyses in human cells and animal disease models. In this study, we validate in human and murine cells a qPCR system for the single-tube assessment of lentiviral vector copy numbers that is suitable for analyses in at least 33 different mammalian species, including human and other primates, mouse, pig, cat and domestic ruminants. The established assay combines the accuracy of single-tube quantitation by duplex qPCR with the convenience of one-off assay optimisation for cross-species analyses and with the direct comparability of lentiviral transduction efficiencies in different species.
authors.affiliation: Cyprus School of Molecular Medicine, Nicosia, Cyprus.
authors.forename: I
authors.initials: I
authors.lastname: Christodoulou
authors.affiliation: Department of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.|King's College London, Gene Expression and Therapy Group London, UK.
authors.forename: P
authors.initials: P
authors.lastname: Patsali
authors.affiliation: Department of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.|King's College London, Gene Expression and Therapy Group London, UK.
authors.forename: C
authors.initials: C
authors.lastname: Stephanou
authors.affiliation: King's College London, Gene Expression and Therapy Group London, UK.
authors.forename: M
authors.initials: M
authors.lastname: Antoniou
authors.affiliation: Cyprus School of Molecular Medicine, Nicosia, Cyprus.|Department of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
authors.forename: M
authors.initials: M
authors.lastname: Kleanthous
authors.affiliation: Cyprus School of Molecular Medicine, Nicosia, Cyprus.|Department of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus.
authors.forename: C W
authors.initials: CW
authors.lastname: Lederer
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008322:Mammals / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction; D016415:Sequence Alignment; D014161:Transduction, Genetic