Published by AAAS finger nestles into the DNA helix at a specific set of three bases (such as GCG), allowing a transcription factor to turn on a specific gene. Klug’s lab and others next showed that they can mix and match different zinc fingers to latch onto specific sequences of DNA—there are 64 possible three-base combinations. Researchers then began exploiting zinc fingers to ferry molecules to a unique position along a chromosome—for example, fusing them to proteins that turn genes on or off so that such proteins would regulate a specific gene. And that inspired the idea of zinc finger nucleases as a way to spur homologous recombination. The strategy is to attach zinc fingers to enzymes called endonucleases that make double strand breaks in DNA. When these enzymes are added to a cell, the usual rate of homologous recombination—1 in a million cells—rises to at least 1 in 1000. In 1996, Srinivasan Chandrasegaran’s group at Johns Hopkins University in Baltimore, Maryland, reported that by attaching three different zinc fingers to these DNA-snipping enzymes, they could cut a piece of a freefloating DNA at a precise location.(The researchers add two nucleases that first land on each side of the point they wish to cut and then combine to snip the DNA.) With Dana Carroll’s group at the University of Utah, Salt Lake City, they later showed that when new DNA was inserted into frog eggs and cleaved by a zinc finger nuclease, the cells then fixed the break. The next step was to see whether zinc finger nucleases could alter specific genes in a cell’s chromosomes. In 2002, Carroll’s group showed in fruit fly larvae that the nucleases could mutate a gene that controls the insect’s color. Some of the resulting flies had patches of yellow where they would normally be dark. That work didn’t attempt to replace the cleaved portion of the color gene, but Carroll’s team reported doing that in 2003 in Science (2 May 2003, p. 764). In addition to the zinc finger nucleases, they added copies of a different version of the color gene into the fly larvae and showed that the larvae incorporated that variant via homologous recombination. In the same issue, Porteus and David Baltimore of the California Institute of Technology in Pasadena reported a similar success. They showed for the first time in human cells that zinc finger nucleases could be used to repair a mutation in the gene, albeit a nonhuman reporter gene inserted into the cells. The first proof of principle that zinc finger nucleases can correct a human disease gene came this spring. In the April online edition of Nature, Porteus and scientists at Sangamo BioSciences Inc. in Richmond, California, showed that such nucleases could make a one-base change in a functional copy of IL2Rγ, the gene that causes X-SCID, in human cells. The zinc finger nucleases worked with relatively high efficiency—18% in primary blood cells and 5% in T cells, the cells that would need to be targeted in X-SCID patients.

Sangamo also intends to use zinc finger nucleases to correct mutations in other blood diseases, such as hemophilia. The general strategy is to isolate bone marrow or other blood-forming stem cells from a patient, correct the mutation in the cells in lab dishes, and put the stem cells back. And in a twist on repairing disease genes, Sangamo is also testing whether zinc finger nucleases can treat HIV patients by disabling the gene for a protein, called CCR5, that the HIV virus uses to enter cells. In 2006, Sangamo and collaborators hope to begin clinical trials in which a person’s HIV-susceptible immune cells would be replaced with bone marrow cells that have had their CCR5 genes knocked out.