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.
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.
Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α1-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α1-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.