Progress and prospects: zinc-finger nucleases as gene therapy agents

Abstract

Zinc-finger nucleases (ZFNs) are powerful tools for experimental gene manipulation. A number of recent papers have shown how this technology can be applied effectively to models of human gene therapy. Significant target genes and useful methods of ZFN delivery have been reported. Important strides have been made in minimizing toxic side effects observed with some ZFNs, which bodes well for their ultimate safety. New tools are available for the design and testing of ZFNs for new target genes. Applications of ZFNs to stem cells have been described, and genuine gene therapy trials appear to be on the immediate horizon.

Figure 1. ZFN binding and activity

The basic arrangement of ZFNs relative to their target DNA (light blue bar) is shown in color. Each ZFN (ZFNa, ZFNb) consists of a set of 4 zinc fingers, shown as smaller ovals, each in a different color to indicate that each binds a different DNA triplet. Each ZF set is linked to a cleavage domain (larger ovals), which must dimerize to cut DNA. Cleavage of the intended target gene can lead to disruption of its coding sequence by inaccurate repair via nonhomologous end joining (NHEJ). When a homologous donor DNA (dark blue bar) is introduced along with the ZFNs, it can be incorporated at the target by homologous recombination (HR). Cleavage at unintended, off-target sites can be toxic to cells and lead to off-target mutations (purple). Some of the approaches to limiting such deleterious effects are described in the text and include modification of the cleavage domain dimer interface and introducing additional fingers to enhance specificity (green).

Figure 2. Scheme for correcting gene sequences downstream of the ZFN cleavage site

A hypothetical target gene is illustrated with 6 exons (boxes 1–6). The sixth exon has a polyA addition sequence that defines the end of the mRNA from this gene. The specific target for ZFN cleavage is envisioned in exon 3. A donor DNA carries sequences of exon 3 and all downstream exons, but without downstream introns. The ends of the donor have homology to the left and right sides of the ZFN target, as illustrated with dashed lines. After cleavage by the ZFNs and HR using those homologies, the target will have the structure illustrated at the bottom. As long as the sequences in the donor are all normal, any mutations in exons 3–6 will be corrected in the eventual mRNA. For example, a mutation in exon 4 of the target (shown as an x) would not be present in the mRNA after incorporation of the donor as shown. Generalized from Lombardo et al..