Optimizing Zinc Finger Nucleases for Use in Mammalian Cells




Pruett-Miller, Shondra M.

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Homologous recombination is a well-established technique that has been used to manipulate the genomes of multiple model organisms with great precision and is therefore being explored as a potential way of performing gene therapy. However, the spontaneous rate of homologous recombination in human cells is too low (10-6) to be therapeutically useful. The most powerful way of stimulating homologous recombination is by introducing a double strand break within the target locus. Zinc Finger Nucleases (ZFNs) are designed proteins that fuse a zinc finger DNA binding domain to the nuclease domain from the FokI restriction endonuclease and have been used to induce double strand breaks at precise sequences. Although ZFNs have been successfully used to stimulate gene targeting at specific loci, several issues remain. First, a generalized optimal design strategy for making effective and safe ZFNs has yet to be established. Second, a systematic evaluation method needs to be established in which novel ZFNs are evaluated for both functionality and safety. We compare the gene targeting efficiencies and cytotoxicity of ZFNs made by the two established design strategies: modular assembly and a bacterial 2-hybrid selection strategy. We have found that ZFNs made by the bacterial 2-hybrid strategy are both more efficient at stimulating gene targeting and less toxic than ZFNs made by modular assembly. We have also found that ZFNs made via the bacterial 2-hybrid strategy are more efficient at gene targeting using a GFP reporter assay and show less cytotoxicity than previously published 4-finger proteins. We also present a generalized strategy for systematically evaluating new ZFNs, which includes a bacterial (-galactosidase transcription assay, a mammalian gene targeting assay, a mammalian flow cytometry based survival assay, and a 53BP1 foci formation assay. These assays provide a standard for future ZFN design and evaluation, particularly those that may be destined for therapeutic use. Because the issue of toxicity is such and important one, we have also developed possible strategies to reduce toxicity of ZFNs. We will discuss two strategies for regulating ZFN protein expression using small molecules. We show that by regulating protein expression to create ZFNs with shortened half-lives, we can maintain high rates of ZFN mediated gene targeting while reducing ZFN toxicity.

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