A new study demonstrates how patient-derived stem cells might one day be used to treat genetic diseases. Scientists from Cambridge, England, corrected a genetic error in stem cells derived from patients with a liver disease, and then differentiated them into liver cells. When injected into the livers of mice, the cells integrated into the organ and started functioning normally.
“They showed that the cells are able to function; that’s a very big achievement and a great opportunity,” says Anil Dhawan, a researcher at King’s College London who was not involved in the study. The study was published today in Nature.
The research combines several techniques, including cell reprogramming and gene editing, that scientists hope will make gene therapy and cell replacement therapies a reality. While safety testing is needed before the treatment can be tested in people, researchers used technologies that left the cells “pristine,” with no signs of the genetic manipulation that took place. This makes them more likely to be suitable for patients.
Researchers first collected cells from patients with α1-antitrypsin deficiency, an inherited disease that strikes one in 2,000 people of northern European descent. People with the disease have a single letter mutation in both copies of the α1-antitrypsin gene. The mutated protein builds up in liver cells, killing off the tissue and eventually necessitating a liver transplant.
Organs for transplant are scarce, so Allan Bradley, a geneticist at the Wellcome Trust Sanger Institute, and collaborators set out to create a new source of replacement tissue. “The liver is a good place to do cell correction therapy because it is a relatively simple organ compared to the brain, lung, or heart,” he says. “It will regenerate to the same size and shape of the original.”
Bradley’s team transformed the adult cells into stem cells using a technique called induced pluripotent stem (iPS) cell reprogramming, in which scientists use a cocktail of genetic factors to turn back the cells’ developmental clock. The resulting cells can both grow more of themselves and can differentiate into any type of tissue.
The benefit of iPS cells is that they are genetically matched to the donor, meaning they can be transplanted back into the patient without triggering an immune attack. (People who receive organ or cell transplants must take immunosuppressive drugs to stop the body from attacking the foreign tissue.)
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.