Abstract

Therapeutic genome engineering is a hallmark of gene therapy but only recent technological advances have permitted the modification of complex genomes in a targeted fashion. Zinc-finger nucleases (ZFNs) have developed into a major playmaker in the genome engineering field and have been employed to trigger the targeted editing of genomes at over 50 gene loci in 11 model organisms, including fruitfly, zebrafish and rat, with allelic frequencies reaching the double digit percentage range. Moreover, ZFN-mediated genome surgery in primary human cells has become a reality and two phase I clinical trials aiming at knocking out the CCR5 receptor in T cells isolated from HIV patients to protect these cells from infection with the virus have been initiated. Considering that specificity is closely linked to ZFN activity and ZFNassociated toxicity, this parameter has been and will be a key quality in any therapeutic application of the designer nucleases. This review summarizes the technological innovations that have successfully catapulted ZFNs into the genome engineering arena and provides an overview of the current state of the art of these nucleases with reference to human gene therapy.

Keywords: Cytotoxicity, gene disruption, gene targeting, genome engineering, genotoxicity, off-target activity, protein engineering, zinc-finger nuclease, Zinc-finger nucleases, CCR5 receptor, T cells, HIV patients, nucleases, DNA-binding domain, immunosorbent assays, -helix, FokI endonuclease, cytometry, phosphorylated, human embryonic stem cells, human immunodeficiency virus, immunotoxicity, C-C chemokine receptor, hemoglobinuria