title: Development of a sensitive assay for detection of replication-competent recombinant lentivirus in large-scale HIV-based vector preparations.
abstract: Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient ex vivo and in vivo gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be demonstrated that lentiviral vector preparations are safe and not contaminated by replication-competent recombinants related to the parental pathogenic virus. Here we describe a sensitive assay for the detection of replication-competent lentiviruses (RCL) in large-scale preparations of HIV-based lentiviral vectors. This RCL assay for lentiviral vectors is based on the principles used for retroviral vectors, using a highly permissive cell line, C8166-45, for RCL amplification and an appropriate positive control virus to establish the assay sensitivity. The assay is capable of detecting 1 RCL infectious unit in a background of 2.5 x 10(8) transducing units of vector in a single test culture. Statistically representative samples from large-scale lentiviral vector productions were assayed using multiple test cultures for each lot. Overall, a total of 1.4 x 10(10) transducing units of vector from 10 independent 14-liter production lots were screened and no RCL was detected. We propose to implement this assay as a release testing for clinical-grade lentiviral vector preparations intended for gene therapy clinical trials.
authors.affiliation: Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, California 94080, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Escarpe
authors.forename: Nathalie
authors.initials: N
authors.lastname: Zayek
authors.forename: Peggy
authors.initials: P
authors.lastname: Chin
authors.forename: Flavia
authors.initials: F
authors.lastname: Borellini
authors.forename: Romain
authors.initials: R
authors.lastname: Zufferey
authors.forename: Gabor
authors.initials: G
authors.lastname: Veres
authors.forename: Veronique
authors.initials: V
authors.lastname: Kiermer
mesh_terms: D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*; D006678:HIV / Q000235:genetics* / Q000254:growth & development*; D006801:Humans; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors; D014779:Virus Replication