title: A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors.
abstract: Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.
authors.affiliation: Oxford BioMedica (UK) Ltd, Medawar Centre, The Oxford Science Park, Oxford, UK. j.miskin@oxfordbiomedica.co.uk
authors.forename: J
authors.initials: J
authors.lastname: Miskin
authors.forename: D
authors.initials: D
authors.lastname: Chipchase
authors.forename: J
authors.initials: J
authors.lastname: Rohll
authors.forename: G
authors.initials: G
authors.lastname: Beard
authors.forename: T
authors.initials: T
authors.lastname: Wardell
authors.forename: D
authors.initials: D
authors.lastname: Angell
authors.forename: H
authors.initials: H
authors.lastname: Roehl
authors.forename: D
authors.initials: D
authors.lastname: Jolly
authors.forename: S
authors.initials: S
authors.lastname: Kingsman
authors.forename: K
authors.initials: K
authors.lastname: Mitrophanous
mesh_terms: D001681:Biological Assay; D002460:Cell Line; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D009052:Leukemia Virus, Murine / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D011995:Recombination, Genetic; D014779:Virus Replication / Q000235:genetics
chemical_list: D012194:RNA-Directed DNA Polymerase