The good news about gene targeting is that it has revolutionized biomedical research. According to the Nobel Committee, which awarded the Nobel Prize to its inventors Mario Capecchi, Martin Evans and Oliver Smithies in October, "Almost every aspect of mammalian physiology can be studied by gene targeting. . . Gene targeting has now been used by so many research groups and in so many contexts that it is impossible to make a brief summary of the results." (See BioWorld Today, Oct. 11, 2007, and Oct. 15, 2007.)

The bad news is that up to now, gene targeting basically has worked only in mice. For reasons that are poorly understood, "mouse embryonic stem cells turn out to be rather different than essentially all other cells," Philip Gregory told BioWorld Today.

Specifically, the frequency of homologous recombination - the switching out of the cell's own DNA for a copy edited by the researcher - is much higher in mouse embryonic stem cells than in other cell types.

In a paper published in the Oct 28, 2007, early online edition of Nature Biotechnology, Gregory, who is vice president of research at Richmond, Calif.- based Sangamo Biosciences, and his colleagues at Sangamo and academic collaborators including Prof. Luigi Naldini at the San Raffaele Telethon Institute for Gene Therapy, Milan, Italy, reported that by using zinc-finger nucleases and a novel gene delivery vector, they were able to apply gene targeting to different cell types, including human stem cells, and achieve gene editing at frequencies that are orders of magnitude greater than what can be achieved with classical methods.

Sangamo uses engineered zinc finger proteins, which are a natural class of transcription factors, for gene editing by correction, disruption or addition of the targeted gene.

In the Nature Biotechnology paper, the scientists achieved efficient gene targeting by using a system with three components: two zinc finger proteins linked to an enzyme that induces double-stranded DNA breaks, and the desired version of the targeted gene, all delivered to the cell via an integrase-deficient lentiviral vector. All of which, once the gene targeting has been accomplished, ultimately are degraded by the cell.

Using that system, the researchers first modified the gene for the interleukin-2 gamma receptor (IL2Rgamma), which is nonfunctional in X-SCID. They found that in different cell types, anywhere from 13 percent to 39 percent of cells had a modified IL2Rgamma gene. It also was possible to add a transgene to the IL2Rgamma site, with an efficiency of up to 6 percent of cells depending on the cell type.

Further experiments showed that by controlling the binding specificity of zinc finger nuclease they used and utilizing an appropriately targeted repair template, the researchers could control precisely where in the genome the DNA sequence would be edited, whether through correction, deletion or addition of genes.

The Nature Biotechnology paper follows a 2005 publication in Nature where the authors demonstrated gene correction in immortalized cell lines or primary human T cells. But the new paper, Gregory said, demonstrated that the technique can achieve the same sort of precise gene editing in "a broad range of cell types including human embryonic stem cells. . . . It opens up tremendous applications for the technology."

The most immediate of those applications are in the area of tailoring cell lines for biomedical research. Sangamo has a collaboration with Sigma-Aldrich to "enable access to the technology from a research reagents perspective."

Gregory said that the science also has potential for applications where human stem cells are being considered, though those applications are much further in the future. The rights to therapeutic applications are exclusively Sangamo's.

The company is working with academic partners on applications in cancer and infectious disease.

Lastly, there may be applications on the farm. Sangamo has a collaboration with Dow Agrosciences for use of the technology to genetically modify maize.

Gregory said that plants "have exactly the same problem" as human cells do - a very low frequency of homologous recombination when using traditional methods.