iPS cells have not yet been tested in humans, in part because the methods typically used to create them permanently alter the cells’ genome. However, in this case, researchers used an iPS method in which the reprogramming factors were expressed only transiently.
To correct the genetic defect that causes the disease, researchers used zinc finger nucleases, enzymes that have been engineered to edit the genome in a very precise manner. The enzymes bind to a specific part of the genome on either side of the area to be corrected and make a snip in the middle. A replacement piece of DNA, delivered along with the nucleases, is then swapped in for the faulty piece.
Researchers added a marker to the replacement DNA that allowed them to pick out the cells that have been repaired. That marker was then removed using another enzyme, leaving the cell free of signs of genetic tampering.
Bradley says the zinc finger approach is more efficient than other methods of genetically engineering cells. They were able to correct a single copy of the gene in about 50 percent of cells, and both copies of the gene in about 5 percent.
While this technology is being broadly used in research, it has only just made the leap into clinical testing. An HIV therapy using a similar technology is currently being tested in patients.
One concern about zinc finger enzymes is the potential to snip DNA in places other than the intended target. Scientists have been able to lessen this problem by engineering more precisely targeted enzymes. But to make sure that the technology did not significantly alter the engineered cells, Bradley’s team sequenced the protein-coding region of the genome afterward.
“Not only did they show they can make genome alterations that are productive and otherwise invisible, they also tracked what’s going on in cells using high-throughput technology looking for genome sequence changes,” says Dana Carroll, a biologist at the University of Utah, who was not involved in the study.
While the researchers did find some genetic changes, none of them seemed to be a consequence of the zinc fingers. They may have been the result of the reprogramming process, or may have been present in the original donor cells. “In this situation, it seems as if the zinc finger nucleases are safe reagents,” says Carroll.