Using precision DNA-cleaving enzymes called zinc finger nucleases (ZFNs) to replace a dysfunctional gene in vivo, researchers successfully restored nearly normal blood clotting in mice with the human blood disease hemophilia B.

The feat, published online yesterday (June 26) in Nature, represents the first time scientists have been able to use ZFN-enabled "genome editing" to permanently correct the DNA of cells within a living animal, and provides hope that the same technique may one day treat a wide range of human diseases.

"This is a significant extension of the zinc finger nuclease technology that could help human medicine," said Mario Capecchi, a professor of human genetics at the University of Utah and a 2007 Nobel Prize winner for gene-targeting discoveries that made possible today's profusion of genetically customized lab mice. "It opens up a new avenue, working directly within the animal to change cells,...

Until now, genome editing has only been performed in vitro—although its products, living cells with revised genes, have been successfully reinfused into their donors, including humans. At Sangamo BioSciences, for example, researchers have used the technique to excise a cellular receptor HIV needs to invade T cells, thus providing HIV/AIDS patients with a reservoir of uninfectable immune fighters—a therapy now in Phase II clinical trials. The advantage of such procedures, said Capecchi, is that "you have the possibility of pulling out the cell that’s working correctly and amplifying that particular copy." But in most organ systems, researchers don't yet know how to extract and return cells without harming the patient, said Katherine High, director of the Center for Cellular and Molecular Therapeutics at the Children’s Hospital of Philadelphia.

Working in vivo has limitations of its own, however. In 2009, High co-led a clinical trial that enrolled 12 legally blind children and adults with a congenital retinopathy. A single injection of a vector-borne corrective gene behind the retina restored partial sight to all 12 patients, most dramatically to the children—an experience High describes as "almost Biblical." But vector-borne genes do not permanently integrate into the cell’s DNA, and thus cannot be passed on to daughter cells, meaning such successes can only be sustained in tissues like the retina where cells don't divide.

To try to achieve a permanent correction of the genome itself, High turned to ZFN technology, which enables researchers to target and precisely cleave specific regions of DNA. The break induces DNA repair enzymes to mend the genetic code, but by offering a template of a desired correction or revision, researchers can effectively trick the cell's own repair mechanisms into inserting the new code in the place of the defective gene. Getting the cell itself to do the job is optimal, because the replacement gene will come "under the control of the normal regulatory elements" that ensure it will work as it should, High explains.

Teaming up with Sangamo, which dubs itself "the zinc finger company," High and her collaborators created mice with a human genetic defect that causes hemophilia B—an inherited bleeding disorder characterized by extremely low levels (less than 1 percent of normal) of clotting factor IX, a protein necessary for coagulation. The researchers then designed a ZFN to cleave the front end of the dysfunctional hF9 gene, and used a viral vector to carry the enzyme to the mice's liver, where factor IX is made. They injected the ZFN, along with a separate vector-borne template of the normal gene's code, into the abdomens of two-day-old mice. When the mice were five weeks old, the team began testing for human factor IX in the plasma of the treated group and controls.

“I didn’t think it would work,” High admits. And when grad student Hojun Li first shared the positive results, postdoc Virginia Haurigot insisted on running the samples again. But it was no fluke: the treated mice showed factor IX levels as high as 6 to 7 percent of normal—high enough that their blood clotted in near normal time.

Furthermore, when the researchers removed part of the 10-week-old mice's livers, triggering regeneration by cell division, their factor IX levels did not drop, and were still going strong at 30 weeks. By contrast, in mice that had received the corrective gene without the ZFN, factor IX levels went up at first, but crashed to near zero after cell division, confirming that the addition of the ZFN had resulted in actual correction of the genome.

Although human trials are at least some time off, the hemophilia treatment is now being tested in dogs, and the study bodes well for "a series of diseases where a small change in the level of protein present will make a big difference," said Capecchi.  While treated mice showed no ill effects, the hurdle in humans will be safety. Cutting DNA, Capecchi cautioned, "is a very reactive set of events that can lead to bad things happening, so it's very important that they get the break where they want it and nowhere else."

H. Li et al., “In vivo genome editing restores haemostasis in a mouse model of haemophilia,” Nature, doi:10.1038/nature10177, 2011.

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