Attenuation of zinc finger nuclease toxicity by small-molecule regulation of protein levels.
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.
Pruett-Miller
Shondra M
SM
Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.
Reading
David W
DW
Porter
Shaina N
SN
Porteus
Matthew H
MH
D002460:Cell Line; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D005786:Gene Expression Regulation / Q000187:drug effects*; D018390:Gene Targeting; D006801:Humans; D061988:Proteasome Inhibitors; D015202:Protein Engineering; D054852:Small Molecule Libraries / Q000494:pharmacology*; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
D061988:Proteasome Inhibitors; D054852:Small Molecule Libraries; D025801:Ubiquitin; D003851:Deoxyribonucleases; D022061:Tacrolimus Binding Protein 1A