Attenuation of Zinc Finger Nuclease Toxicity by Small-Molecule Regulation of Protein Levels

Abstract

Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity. Zinc finger nucleases (ZFNs) are a powerful tool to create site-specific genomic modifications in a wide variety of cell types and organisms and are about to enter human gene therapy clinical trials. An important aspect of using ZFNs for use in gene therapy is to minimize off-target effects. We made ZFNs that contain destabilizing domains on their amino-terminus. The expression level of the modified ZFNs could be increased transiently by the addition of a small molecule, either a proteasome inhibitor or Shield1. We demonstrate that off-target effects can be reduced without compromising gene targeting efficiency by using small molecules to limit the maximal expression of the ZFNs to a narrow window. The ability to regulate ZFN expression using small molecules provides a new strategy to minimizing off-target effects of ZFNs and may be an important way of ultimately using ZFNs for clinical use in gene therapy protocols.