title: Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors.
abstract: It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.
authors.affiliation: Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Farley
authors.affiliation: Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
authors.forename: Laura
authors.initials: L
authors.lastname: McCloskey
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Thorne
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Semih U
authors.initials: SU
authors.lastname: Tareen
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Nicolai
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Campbell
authors.affiliation: Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
authors.forename: Richard
authors.initials: R
authors.lastname: Bannister
authors.affiliation: Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
authors.forename: Hannah J
authors.initials: HJ
authors.lastname: Stewart
authors.affiliation: Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
authors.forename: Laura Je
authors.initials: LJ
authors.lastname: Pearson
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Bentley J
authors.initials: BJ
authors.lastname: Moyer
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Scott H
authors.initials: SH
authors.lastname: Robbins
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Leah
authors.initials: L
authors.lastname: Zielinski
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Tae
authors.initials: T
authors.lastname: Kim
authors.affiliation: Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
authors.forename: Pippa A
authors.initials: PA
authors.lastname: Radcliffe
authors.affiliation: Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
authors.forename: Kyriacos A
authors.initials: KA
authors.lastname: Mitrophanous
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Gombotz
authors.affiliation: Oxford BioMedica (UK) Limited, Windrush Court, Transport Way , Oxford, UK.
authors.forename: James E
authors.initials: JE
authors.lastname: Miskin
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Brenna
authors.initials: B
authors.lastname: Kelley-Clarke