Cell-free selection of DNA-binding proteins for future gene therapy applications

Engineered DNA-binding proteins, in particular zinc finger proteins (ZFPs), have broad-ranging applications in gene therapy. An engineered ZFP transcription activator targeted to the VEGF locus is currently undergoing clinical trials for the induction of angiogenesis. A number of ZFP gene switches have been developed which allow highly controllable regulation of therapeutic transgene expression based on small molecule inducers/repressors. Finally, engineered ZFP nucleases have been used to correct a gene sequence in a living cell by stimulating homologous DNA recombination, suggesting a new, highly targeted approach to gene therapy. All these approaches rely on DNA-binding protein engineering, which in the past has mainly been achieved by selection using phage display. However, a convenient cell-free selection method known as in vitro compartmentalization (IVC) has previously been used to engineer DNA-binding proteins with enzymatic activities (e.g. polymerase and methylase), and the method has recently been extended to the engineering of sequence-specific ZFP DNA-binders. Below we describe the IVC procedure and review the progress made in applying this to the problem of facilitating the engineering of DNA-binding proteins.