Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rgamma gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

Zinc-finger proteins (ZFPs) that recognize novel DNA sequences are the basis of a powerful technology platform with many uses in drug discovery and therapeutics. These proteins have been used as the DNA-binding domains of novel transcription factors (ZFP TFs), which are useful for validating genes as drug targets and for engineering cell lines for small-molecule screening and protein production. Recently, they have also been used as a basis for novel human therapeutics. Most of our advances in the design and application of these ZFP TFs rely on our ability to engineer ZFPs that bind short stretches of DNA (typically 9-18 base pairs) located within the promoters of target genes. Here, we summarize the methods used to design these DNA-binding domains, explain how they are incorporated into novel transcription factors (and other useful molecules) and describe some key applications in drug discovery.

We have targeted the activation of an endogenous chromosomal locus including the human erythropoietin gene using synthetic transcription factors. These transcription factors are targeted to particular DNA sequences in the 5-flanking region of the erythropoietin gene through engineering of a zinc finger DNA binding domain. The DNA binding domain is linked to a VP16 transcriptional activation domain. We find that these synthetic transcription factors invariably activate transiently transfected templates in which sequences within the 5 flank of the erythropoietin gene are fused to a luciferase reporter. The efficiency of activation under these circumstances at a defined site is dependent on DNA binding affinity. In contrast, only a subset of these same zinc finger proteins is able to activate the endogenous chromosomal locus. The activity of these proteins is influenced by their capacity to gain access to their recognition elements within the chromatin infrastructure. Zinc finger transcription factors will provide a powerful tool to probe the determinants of chromatin accessibility and remodeling within endogenous chromosomal loci.The enormous progress in our understanding of gene control in eukaryotes using model systems presents substantial opportunities to apply this knowledge for therapeutic benefit in man. The rational design and engineering of components of the transcriptional machinery provide a powerful means to test conventional paradigms for the roles of protein-DNA and proteinprotein interactions in gene regulation. These designer transcription factors may also provide novel means of regulating endogenous chromosomal loci for a variety of beneficial purposes. Over the past decade, the primary structural determinants of DNA recognition by zinc fingers of the Cys 2 -His 2 type have been elucidated (1-8). Designer transcriptional regulators containing three or more zinc finger domains have been used in isolation (9, 10) or following linkage to transcriptional activation (9, 11-13) or repression domains (12, 13). These novel proteins control the transcription of reporter genes both transiently transfected into human cells (9, 11-13) and endogenous chromosomal loci (9, 13). Exactly how these regulatory functions are exerted remains to be resolved. An important issue in considering transcription factor function in eukaryotes is the capacity of the regulator to gain access to specific sites in chromatin and recruit transcriptional co-activators and co-repressors that modify the chromatin environment (14). These issues have been investigated for the archetypal Cys 2 -His 2 zinc finger protein and transcriptional regulator TFIIIA 1 (15-26). There is general agreement that the nucleosome can impede recognition of specific promoter elements by TFIIIA (15,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) and that modification of histone-DNA interactions through nucleosome repositioning (18,19), histone depletion (20, 21), and removal of the histone tails (22, 23) can promote TFIIIA binding to a nucleosomal infrastructure. Ac...