Abstract 5919: Component-specific qPCR assays for characterization and identity testing of multigenome ZVex®, a dendritic cell-targeting lentiviral vector platform
Authors: Bajaj Anshika , Lin Tsai-Yu , Ngo Lisa Y. , Murphy Michele , Kelley-Clarke Brenna , Gombotz Wayne R. , Meulen Jan H. ter , Berglund Peter 
Published: August 2018

Abstract
Background: ZVex is a novel, integration-deficient, dendritic cell-targeting lentiviral vector platform currently being evaluated in clinical trials in sarcoma patients. The vector is capable of delivering multiple full-length genes simultaneously. Here, we describe ZVex preparations encoding the human cancer testis antigens MAGE-A1, 3, 4, 10 and Interleukin 12. Because lentiviral vectors package two RNA molecules each, this ZVex approach results in the generation of 15 possible permutations of homozygous or heterozygous vector genotypes. The homologous nature of the MAGEA family members (50-80% sequence identity) poses significant challenges to their specific detection and quantification in a MAGEA gene pool. Here, we describe highly specific qPCR assays for detection of the 4 MAGEA genes and IL12 for characterization and identity testing of multigenome ZVex preparations.
Materials and Methods: Genes were codon-modified to enable specific detection of vector-expressed genes over cellular sequences. The entire IL12B and short, non-overlapping, divergent target regions in each of the 4 MAGEA genes were carefully selected as templates for primer/probe design. Primer-blast was used to predict absence of off-target detection of cross-reacting genomic sequences. To further enhance the specificity of the primers/probes to each of their target MAGEA, potential off-target amplicons that can be generated in the other 3 MAGEA genes due to base pair mismatch were further sequence-modified to reduce sequence identities. ZVex vectors were generated by transfecting 4 or 5 different plasmids encoding the codon modified full-length MAGEA and/or IL12 genes in a producer cell line along with essential vector packaging plasmids. Single/multiplex qPCR assays were performed to analyze vector RNA/reverse-transcribed DNA.
Results: The primer/probe combinations were highly specific for each of the 4 MAGEA genes and IL12 as indicated by melt curve analysis and producing a single qPCR product at the expected size. The primer/probes specifically detected from 10 to 107 copies of their target MAGEA with &gt;=93% efficiency and specifically detected reverse-transcribed vector DNA in a cell-based vector transduction assay. The assays demonstrated relative gene specific titers that correlate with transfected plasmid ratios. MAGEA/IL12 specific qPCR assays benchmark well against previously established “gold standard” qPCR assays used for quantifying total ZVex vector genome titers.
Conclusions: Component-specific qPCR assays were developed for the specific assessment of constituent genes in multigenome ZVex preparations. These assays demonstrate inter/intragene specificity, are amenable to multiplexing, and can be used for quality control, titration, characterization, and identity testing of ZVex lots in a cGMP setting.
Citation Format: Anshika Bajaj, Tsai-Yu Lin, Lisa Y. Ngo, Michele Murphy, Brenna Kelley-Clarke, Wayne R. Gombotz, Jan H. ter Meulen, Peter Berglund. Component-specific qPCR assays for characterization and identity testing of multigenome ZVex®, a dendritic cell-targeting lentiviral vector platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5919.