title: Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates.
abstract: Using lentiviral vector products in clinical applications requires an accurate method for measuring transduction titer. For vectors lacking a marker gene, quantitative polymerase chain reaction is used to evaluate the number of vector DNA copies in transduced target cells, from which a transduction titer is calculated. Immune Design previously described an integration-deficient lentiviral vector pseudotyped with a modified Sindbis virus envelope for use in cancer immunotherapy (VP02, of the ZVex platform). Standard protocols for titering integration-competent lentiviral vectors employ commercial spin columns to purify vector DNA from transduced cells, but such columns are not optimized for isolation of extrachromosomal (nonintegrated) DNA. Here, we describe a 96-well transduction titer assay in which DNA extraction is performed in situ in the transduction plate, yielding quantitative recovery of extrachromosomal DNA. Vector titers measured by this method were higher than when commercial spin columns were used for DNA isolation. Evaluation of the method's specificity, linear range, and precision demonstrate that it is suitable for use as a lot release assay to support clinical trials with VP02. Finally, the method is compatible with titering both integrating and nonintegrating lentiviral vectors, suggesting that it may be used to evaluate the transduction titer for any lentiviral vector.
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Michele E
authors.initials: ME
authors.lastname: Murphy
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Chintan D
authors.initials: CD
authors.lastname: Vin
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Megan M
authors.initials: MM
authors.lastname: Slough
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Gombotz
authors.affiliation: Immune Design , Seattle, Washington, USA.
authors.forename: Brenna
authors.initials: B
authors.lastname: Kelley-Clarke