Integrated Multimodal Evaluation of Genotoxicity in ZFN-Modified Primary Human Cells

Abstract

Iatrogenic adverse events in clinical trials of retroviral vector-mediated gene-corrected cells have prioritized the urgent need for more comprehensive and stringent assessment of potentially genotoxic off-target alterations and the biosafety of cells intended for therapeutic applications. Genome editing tools such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 nuclease systems are being investigated as safer and efficient alternatives for site-directed genome modification. Using site-specific integration into the AAVS1 locus of primary human cells as an example, we present an integrated approach to multimodal investigation of off-target alterations and an evaluation of potential genotoxicity induced by ZFN-mediated integration of a therapeutic transgene.

Keywords: AAVS1 site-specific; Cell and gene therapy; Genotoxicity; Off-target; Primary human cells; RNA-seq, bioinformatics; Transgene integration; Whole-genome sequencing; Zinc finger nuclease.